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Characterization of a Novel Amino-Terminal Domain From A Copper Transporting P-Type ATPase Implicated in Human
Genetic Disorders of Copper Metabolism
Michael DiDonato
A thesis submitted in conformity with the requirements for the degree of Doctor o f Philosophy Graduate Department o f Biochemistry
University of Toronto
O Copyright by Michael DiDonato 1999
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This rhesis is dedicated ro the mentor): of
Micltele DiDonato
and
Domenico Beftini
ABSTRACT
Characterization of a novel copper transporting P-type ATPase implicated in human genetic disorders of copper metabolism
Degree of Doctor of Philosophy, 1999.
Michael DiDonato
Graduate Department of Biochemistry, University of Toronto
The -70 kDa copper binding domain fiom the Wilson disease copper transporting P-
tvpe ATPase was expressed and purified as a fiision to giutathione-S-transferase (GST).
Following purification, a detailed study of the metal binding characteristics of the domain, as
well as the stmctural consequences of metal binding, was carried out. In order to determine
the rnetal binding specificity of the domain, immobilized metal ion affinity (IMAC) and
blotting analyses were performed. The results of these analyses indicate that the domain is
able to bind a wide range of transition metals with varying affinities. The apparent order of
affinity was found to be: Cu(I)/Cu(II)>Zn(Li)>Ni(II)>Co(II). Copper bound to the domain is
in the + I oxidation state as assessed using the colorimetric copper(1) chelator bathocuproine
disulfonic acid (BCS) and x-ray absorption near edge structure (XANES) spectroscopy.
Neutron activation analysis (NAA) indicated a coppedprotein stoichiornetry of 6.5 copper
atoms / mole of protein. In competitive " ~ n blotting experirnents non-radioactive copper
(Cu(1) or Cu(I1)) was able to inhibit %nc binding to the domain in a sigmoidal marner,
suggesting the prcsence of cooperativity. This sigmoidal inhibition pattern was not observed
for any of the other metaIs tested. Detailed structural studies using circular dichroism (CD)
spectroscopy indicate that significant changes in both secondary and tertiary structures take
iii
place in the domain upon copper binding. Furthemore, the structural changes which occur
upon the addition of zinc are significantly different fiom those which occur with copper. X-
ray absorption spectroscopy (XAS) indicates that the copper atoms are ligated by two sulfur
atoms in a distorted linear arrangement. The XANES spectra did not change significantly
with incremental addition of copper to the domain indicating that al1 six binding sites may be
stnrcturally sirnilar.
No amount of experimentation can prove me right; a single experiment can prove me wrong.
-Albert Einstein
Acknowledgments
I would like to thank al1 the people who, in one way or another, have helped me in my
graduate career over the past five years. First and foremost I would like to thank my
supervisor Dr. Bibudhendra Sarkar for allowing me the freedom to pursue my interests in his
lab and for al1 his help and guidance over the years. 1 would also like to thank al1 the
members of my cornmittee, Dr. David Mademan, Dr. Robert Moms and Dr. Diane Cox for
al1 their suggestions, help and encouragement. I could not have reached this point without
the help and assistance of my Iab-mates both past and present. To Suree, thank you for
always bcing there as a friend and for your constant Stream of suggestions and solutions to
rny problerns, you have been a tnie role model. Many thanks to Tokameh who shared with
me both the pain and exhilaration of research during Our tour of duty in the lab. you were
missed over the last year. 1 would also like to thank Nira and Loretta for al1 their help and
assistance over the years. To al1 the members of the Sarkar Lab (past and present) Suree,
Tokameh, Mike, Negah, Lakshmi, Andrew, Damiano, Xuefeng and Jin-you, thank you for
making the lab a great place to work. 1 wil1 miss those lab lunches and the crosswords.
1 must also take this opportunity to thank the person who has had the most influence
on my life, my wife Grace DeSantis. She has helped in more ways than 1 can say and has
becn the rock which has anchored me in the Storm. She has always been there with her
support and bas never tired of hearing my depressing research stories. Her intelligence and
perseverance has always been an inspiration to me.
1 would also like to thank al1 rny fnends for their help, support and friendship over the
years. Thank you to Nana and Natalie first for teaching me molecular biology and for
making the Pulleyblank lab a great place, but more importantly for your fi-iendship and
support through the "rough" times in grad school. To Voula, thanks for al1 the QC on my
data presentation (it really paid off in Edmonton!), for cirafting me into the BGSU and for
enlightening me on the "purpose" of glycerïn soap. To Dina, thanks for al1 the spanakopita
and dolmathakia and for always having interesting stones to tell me. To Randy, thanks for
getting me involved with Aliquotes and giving me an excuse to put up a web site, it was great
whils it lasted.
To everyone else on the third floor who I've missed, thanks for making rny stay at
Sick Kids interesting and fun, you will al1 be missed. Lastly I would like to thank my famiiy
for always supporting my interests and encouraging me to strive for higher goals. I love you
al1 very much.
vii
Table of Contents
ABSTRACT ACKNO WLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABBREVIATIONS
CHAPTER 1
Introduction
1 . Metals in Biological Systems
1.3 Form and Function of Copper in Bmlogical Systems I -2.1 Types of copper 1 2.2 Important copper-containing enzymes in humans
1.3 Copper Homeostasis in Humans 1.3.1 Copper uptake and circulation 1.3 -2 Membrane transport of copper . 1.3.3 Intracellular copper trafficking 1.3.4 Excretionofcopper
1.4 Cation Transporting ATPases 1.4.1 Types of cation transporting ATPases
1.4.1.1 P-type ATPases 1.4.1.2 V-type and F-type ATPases
1.4.2 Meakes and Wilson Disease Copper Transporting ATPases
1.5 Human Genetic Disorders of Copper Metabolism 1 S. 1 Menkes disease
1 -5. 1.1 Genetics of Menkes disease 1 S. 1.2 Clinical and Biochemical findings 1.5.1.3 Currenttreatments 1 .S. 1 -4 Animal models
viii
Table of Contents (cont'd)
1 -5.2 Wilson disease 1 -5.2.1 Genetics of Menkes disease 1 -5.2.2 Clinical and Biochemical findings 1 -5.2.3 Current treatments 1 -5.2.4 Animal models
1.6 Project Rationale
CHAPTER 2
Materials and Metbods
2.1 DNA Preparation 2.1.1 Ce11 lines 2.1 -2 Preparation of competent BL2 1 (DE3) and DH5a Cd1 2.1.3 Transformation of competent BL2 l(DE3) and DHSa CelL
2.1.3.1 Preparation of glyceroI stocks 2.1.4 Purification of plasmid DNA 2.1.5 DNA agarose gel electrophoresis 2.1.6 Purification of DNA restriction fiagrnents from agarose gels 2.1 -7 DNA Sequencing 2.1.8 Restriction enzyme digests and ligation reactions
2.2 Construction of Expression Vectors 2.2.1 Construction of @EX-WCBD 2.2.2 Construction of pGEX-WCBD(P)
2.3 Expression and Purification of GST-WCBD and GST-WCBD(P) 2.3.1 Buffer and reagent preparation 2.3.2 Expression of GST-WCBD and GST-WCBD(P) 2.3.3 Purification of GST-WCBD and GST-WCBD(P) 2.3 -4 SDS-PAGE 2.3.5 Protein quantitation 2.3.6 N-Terminal Amino Acid Sequencing 2.3.7 Arnino Acid Analysis
2.4 Removal of GST Fom GST-WCBD and GST-WCBD(P) 2.4.1 Thrombin cleavage of GST-WCBQ
Table of Contents (cont'd)
2.4.1.1 Optimization of thrombin cleavage conditions 2.4.2 PreScissionm cleavage of GST-WCBD(P)
2.5 Characterization of the Metal Binding Properties of WCBD 2.5.1 Immobilized Meta1 Ion Afinity Chromatography (MAC) 2.5.2 6 5 ~ n 0 Blotting Assay
2.5 -2.1 Effect of pH on 6 S ~ n ( l ~ Blotting Assay 2.5.2.2 Effect of DTT on 6 5 ~ n ( ~ ~ Blotting Assay
2.5.3 Compehtion "z~(II) Blotting Assay 2.5.4 Stoichiometry of metal binding 2.5.5 Neutron Activation Analysis (NAAI
2.6 Characterization of the Copper Binding Sites 2.6.1 Preparation of apo-protein 2.6.2 Preparation of Samples For XAS and CD analysis
2.6.2.1 Preparation of Dry XAS Samples 2.6.2.2 Preparation of Liquid XAS and EPR Samples
2.6.3 EXAFS and XANES Analysis of Cu-GST-WCBD 2.6.4 Oxidative release of copper fiom GST-WCBD
2.7 Conformational Analysis of GST-WCBD and WCBD 2.7.1 Analysis of protein structural changes upon metal binding 2.7.2 Estimation of secondas. structure content
CHAPTER 3
Meta1 Binding Properties of the N-Terminal Copper Binding Domain from the Wilson Disease Copper Transporting P-Type ATPase (WND)
3. L Introduction 105
3.2 Results 1 07 3.2.1 Expression and purification of GST-WCBD, GST-WCBD(P), and
WCBD 107 3 2 . 2 Metal binding charactenstics of GST-WCBD and WCBD 110
3.2.2.1 Immobilized metal ion affinity chrornatograpby (MAC) 110 3.2.2.2 Neutron activation analysis (NAA) 112 3 -2.2.3 6 5 ~ i n c blotting analysis 112
Table of Contents (cont'd)
3 .M.4 Cornpetition 65~?nc blotting analysis
3.3 Surnmary and Discussion
CHAPTER 4
Structural Characterization of the Copper Binding Domain And Its Meta1 Binding Sites
4.1 Introduction
3.2 Results 4.2.1 X-ray absorption spectroscopy (XAS) analysis of GST-WCBD 4.2.2 Circular dichroism analysis of GST-WCBD and WCBD 4.2 -3 Estimation of secondary structure content 4.2.4 Oxidative release of copper from GST-WCBD
4.3 Summary and Discussion 4.3.1 Hypothetical mode1 for the function of the N-terminal copper
binding domain of ATP7B in vivo
CHAPTER 5
General Discussion and Future Directions
5.1 General Discussion
5.2 Future Directions
5.3 Literature Cited
List of Tables
Table 1.1
Table 1.2
TabIe 1.3
Table 1.4
Table 1.5
Table 1.6
Table 1.7
Table
Table
Table 1-10
Table 1.1 1
Table 2.1
Table 4.1
Table 4.2
Function of Some Biologically Important Metal Ions
Physiological Role of Copper-Containing Enzymes in Humans
Developmentally Important Copper-Containing Enzymes in Humans
Dietary Modifiers of Intestinal Copper Uptake in Humans
meta ai Content of Rat Bile
Metal Transporting Proteins Containing N-terminal HMA Domains
Symptoms of Classical Menkes Disease and Occipital Hom Syndrome (OHS)
Typical Copper Measwements in Normal Adults and Menkes Disease Patients
Features of Menkes Disease in Humans and the Mottled Mouse Mutants
Typical Copper Measurements in Normal Adults and Wilson Disease Patients
Cornparison of Biochemical Findings in Wilson Disease and its Animal Models: Toxic Milk Mouse and Bedlington Terriers
CD Parameters Used in the Conformational Analysis of GST-WCBD and WCBD
Secondary Structure Estimation for Metal Titrated WCBD and GST- WCBD Using SELCON and K2D
CD Transitions of Aromatic S ide Chains 141
xii
List of Figures
Figure 1.1
Figure 1.2
Figure 1.3
Figure 1.4
Figure 1.5
Figure 1.6
Figure 1.7
Fi,oure 1.8
Figure 2.1
Figure 2.2
Figure 3.1
Figure 3.3
Figure 3.3
Figure 3.4
Figure 3.5
Figure 3.6
Figure 3.1
Figure 4.2
Figure 4.3
Figure 4.4
Intestinal and Cellular Copper Uptake Pathways
Transport Mechanism of SERCA and Na-/K- ATPases
Topology of Type 1 and Type U ATPases
Cornparison of Wilson (W), Menkes (MNK) and Rat (rWND) Copper Transporting ATPases
Proposed Structure for Wilson and Menkes Copper ATPases
Phylogenetic Relationship Between HMA Containing Proteins
Genomic Structure of the Disease Gene (ATP7A)
Genomic Structure of the Wilson Disease Gene (ATP7B)
Parental and Moditied GST Fusion Expression Vectors
GST-WCBD(P) Expression Vector with PreScissionTM Cleavage Site
Effect of Copper on BL2 1 (DE3) Growth Following Induction
Summary of Typical Purification of WCBD and GST-WCBD
Immobilized Metal Ion Affinity Chromatography (IMAC) of GST- WCBD
EEect of pH on the Binding of
Effect of Reducing Agents on the Binding of 6 5 ~ n
Cornpetition of 6 5 ~ n Binding to WCBD
Normalized Copper XANES Spectra For GST-WCBD with Various Amounts of Copper
EXAFS Spectra For GST-WCBD with Various Amounts of Copper
CD Spectra of GST-WCBD Titrated with Copper
CD Spectra of GST-WCBD Titrated with Zinc
xiii
List of Figures (cont'd)
Figure 4.5 CD Spectra of WCBD Titrated with Copper
Figure 4.6 CD Spectra of WCBD Titrated with Zinc
Figure 4.7 Oxidative Release of Bound Copper From WCBD
Figure 4.8 Hypothetical Mode1 for the Function of WND in vivo
xiv
Abbreviations
p-Me
BCS
BSA
CAPS
cDNA
DNA
DTT
EDTA
EPR
ERDBD
EXAFS
GST
HMA
IPTG
LB
NAA
OMIM
PAGE
PMSF
RT
RT-PCR
SDS
SOC
TAE
TCA
TCEP
WCBD
XANES
?CAS
fi-Mercaptoethanol
Bathocuproinedisulfoaic acid
Bovine serum albumin
3-(cyc1ohexylamino)- 1 -propanesulfonic acid
Cornplimentary deoxyribonucleic acid
Deoxyribonucleic acid
Dithiothreitol
Ethy lenediaminetetraacetic acid
Eiectron paramagnetic resonance
Estrogen receptor DNA binding domain
Extended X-ray absorption fine structure
Glutathione-S-Transferase
Heavy metal associated domain
Isopropyl b-D-Thiogalactopyranoside
Luria-Bertani Broth
Neutron activation analysis
Online Mendelian Inheritance in Man
Polyacrylamide gel electrophoresis
Phenylmethylsulfonyl fluoride
Roorn temperature
Reverse transcription potymerase chain reaction
Sodium dodecyl sulfate
Save Our Cells (medium)
Tris-Acetate-EDTA Buffer
Tnchloroacetic acid
TrÎs(2-carboxyethy1)phosphine
Wilson Disease Copper Binding Dornain
X-ray absorbance near edge structure
X-ray absorption spectroscopy
CHAPTER 1
Introduction
1.1 Metals in Biologieal Systems
Metals play a vital role in the proper hnction of many biological processes and are
therefore integral to the maintenance of life. It is believed by some, that the origin of life
itself was aided in part by the presence of various metals in the primordial environment of the
early Earth (Ochiai, 1995; Osterberg, 1995). The role o f metal ions in biological systems has
evolvcd to include important roles in transport, signaling, catalysis and structure-promotion.
The bioIogical h c t i o n s of some selected metal ions are surnmarized in table 1.1. There are
many attributes of metal ions which make them especially usefiil in biological systems. Two
of the most important are complex formation and redox properties. When metal ions form
complexes, the properties of the metal as well as the ligand are altered. The simplest
cxampIe of this is the alteration of the pK, of water upon the addition of metal. When
iron(II1) is introduced into an aqueous solution the pK, of the bound water ligands is
drarnatically altered and is substantially lower than that of the b u k water, 3.5 versus 15.6
(Burgess, 1978). This property can also be obsewed with other ligands and is integral to the
hnction of proteins which contain a metal factor.
In these cases the metal ion may promote electrophilic activation, charge
neutralization, and the creation of a more effective nucleophile. Alternatively, the metal ion
rnay help establish the proper substrate geornetry, or activate the substrate by imposing a
distortion or strain (Phipps, 1995). In many cases the binding of a metal ion to a protein can
lead to the stabilization of a particular conformation which may be significantly different
than what would be observed in the absence of metal (Glusker, 1991). The redox properties
of certain metal atoms also play an important part in biological systems since the various
oxidation States o f the metal usually have different properties and provide
Table 1.1 Function of Some Biologically Important Metal Ions
Sodium Potassium
Magnesium Calcium
Charge carrier; osmotic balance Charge carrier; osmotic balance
Structure, hydrolase; isomerase Structure, signaling; charge carrier
Vanadium Nitrogen fixation; oxidase
C hromium Involvement in glucose tolerance and rnetabolism
Molybdenum Nitrogen fixation; oxidase; 0x0 transfer
Tungsten Dehydrogenase
Photosynthesis; oxidase: structure Oxidase; dioxygen transport; electron transfer, nitrogen fixation
Cobalt Oxidase; aikyl group transfer
Nickel Hydrogenase ; hydrolase
Copper Oxidase; dioxygen transport; electron transfer
Zinc Structure; hydrolase Adapted tiom (Lippard and Berg, 1994).
an additional control point for modulation of the proteins' activity. Many metalloenzymes
cany out complex redox reactions which are critical to the maintenance of life. These are
usually two electron reactions which ofien involve group transfer as wefl. Examples of these
types of reactions are the addition and removal of oxygen f?om a substrate.
Although a critical component of many enzymes, most metal ions are only required in
trace amount. An excess of metal ionst especially those with redox properties such as copper
and iron, pose a potentiat threat to the organism. As a result, many organisms have had to
evolve specific and efficient methods to regulate both the uptake and efflux of metal ions
from their cells in order to avoid the toxic effects of metal accumulation and the equally toxic
effects of metal deficiency. The result is a whole host of proteins, and control mechanisms
which are only now becoming well understood.
1.2 Form and Function of Copper in Biological Systems
1.2.1 Types of Copper
Copper is an essential element which is present in the active sites of many enzymes
carrying out a variety of fùnctions (Table 1.2). Most of biological copper exists bound to
proteins and these copper binding sites are divided into 3 main types based on their
spectroscopic properties. Type 1 copper is not normally found in humans and consists of a
single copper atoms ligated by four ligands in a distorted tetrahedral arrangement. This type
of copper is characterized by an intense absorption (E = 3000 - 5000 M-' cm-') at - 625 nm,
imparting a blue color to the protein, and an unusually small hyperfine splitting constant in
its EPR spectra (Solomon et al., 1976). The intense blue color of these proteins has led to
Table 1.2 Physiological Role of Copper-Containing Enzymes in Humans
Physiological Role Protein Radical Scavenging CulZn Superoxide Dismutase
Metal Transport
Electron Transport
Ferroxidase Activity
Adenosine and Homoc ysteine Synthesis
Metallothionein Ceruloplasrnin
Metallothionein Cemioplasmin Albumin ATOX I +
c c s 1 +
cox 17+
Cytochrome oxidase
Ceruloplasrnin Ferroxidase II
Adenosy lhomocysteinase
Blood Coagulation Factor V - Factor VI1
' ~ecen t ly identi fied intracellular copper chaperones.
this type of copper also being referred to as 'blue copper'. The type 1 copper center can
accommodate the geometry of both copper(1) and copper(iI). The second type of copper
centers are referred to as 'normal' copper since their spectroscopic properties are similar to
those of rnany low molecular weight copper complexes. This site is characterized by a
hyperfine coupling constant as detected by EPR, which is considerably larger than for type 1
copper and a weak absorption in the W N i s spectrum. Type 2 copper centers usually consist
of a distorted square planar arrangement of ligands and are practically colorless (Harris,
1995). The usual oxidation state for copper in this site is +2. The third type of copper differs
from the first two in that it is a binuclear site. Type 3 copper sites exist as a Cu([)-Cu(1)
couple to which oxygen is able to bind bridging the two copper atoms. The binding of
oxygen as a stable peroxide species results in the oxidation of the copper pair from the +1 to
the +-2 oxidation state (Solomon, 1988). The Cu(I1)-Cu(I1) form of the protein would be
expecied to have an EPR spectrum since Cu(Ii) is a d9 system; however, the proximity of the
two copper atoms (- 3.5 A (Volbeda and Hol, 1988)) results in spin coupling abolishing the
EPR spectra. The oxygenated type 3 copper site is characterized by a strong absorption at
330 nm (Harris, 1995).
Although the copper sites descnbed above are the most common of those found in
bioiogical systems, another class of copper site which is composed of a type 3 and type 2 site
has also been described. This trinuclear site is found in al1 multicopper oxidases described
thus far and is composed of a triangular arrangement of copper atoms with the binuclear type
3 copper site at the base and a type 2 copper site at the apex (Allendorf et al., 1985; Spira-
Solomon el al., 1986). Lastly, mono copper sites based on copper in the +1 oxidation state
have sometimes been referred to as type 4 copper. However, since copper is in the i l
oxidation state, with a dlo closed-shell system, it is spectroscopically inactive and hence not
easily detected. In order to characterize these types of copper centers, techniques such as
magnetic circular dichrosim and various types of XAS must be employed.
1.2.2 Important Cupper Containing Enzymes in Humans
There are many developmentally important enzymes in humans which require copper
to function properly (Table 1.3). -4 deficiency of copper in the body will lead to a decreased
activity of these enzymes with serious consequences. It is this scenario which is present in
Menkes disease, where many of the observed phenotypes can be traced back to a deficiency
in copper-requiring enzymes. Among the most important copper-containing enzymes in
humans are cytochrome c oxidase, ceruloplasmin, lysyl oxidase, and CdZn superoxide
dismutase (SOD).
Ceruloplasmin is a large, secreted, multicopper serum glycoprotein which carries
approximately 95% of al1 copper circulating in the body (Frieden, 1986). The crystal
structure of holoceniloplasmin bas been solved and shows that the holoprotein contains six
copper atoms, two type 1 sites, type 2 and 3 sites and a type 4 site (Zaitseva er al., 1996).
The type 3 and type 2 sites are arranged in trinuclear cluster which is similar to that found in
laccase and imparts ferroxidase activity on the protein (Rydén, 1988). The protein is
synthesized in hepatocytes and copper incorporation takes place early in the secretory
pathway possibly in the golgi or rough endoplasmic reticulum, with N-glycosylation not
being required for copper loading (Sato and Gitlin, 1991). Although the level or availability
of copper has no effect on the synthesis or secretion of the protein, apoceruloplasmin lacking
oxidase activity is unstable and rapidly degraded once secreted (Gitlin et al., 1992). The
Table 1.3 Developmentally 1a i~ ; tûz t Ccnner-Contakg T c Enzymes in Humans
Enzyme Function Consequence of Deficiency
Cytochrome oxidase (EC 1.9.3.1)
Superoxide dismutase (EC 1.15.1.1)
Tyrosinase (EC 1.14.18.1)
Dopamine-f3-hydroxylase (EC 1.14.17.1)
Lysyl oxidase (EC 1.4.3.13)
Electron ttansport Muscle weakness, neurological chah effects, hypothermia
Free radical Implicated in arnyotrophic scavenger lateral sclerosis (ALS)
Melanin synthesis Loss of pigmentation
Catecholamine Neurologie effects production
Cross-linking of Arterial abnormalities, loose collagen and elastin skin and joints
Ferroxidase, possible Anemia, secondary copper (EC 1.16.3.1) copper transporter de ficiency
precise function of ceruloplasmin in the body is not known, although roles in copper
transport (Campbell et al., 198 1 ; Dameron and Harris, 1987; Hilton et al., 1995; Hsieh and
Frieden, 1975; Marceau and Aspin, 1973) and iron metabolism (Frieden, 1986; Hams,
199Sa; Roeser et al., f 970), due to its ferroxidase activity, have been propose&
The trinuclear copper cluster is responsible for activating oxygen during the catalytic
cycle of the protein and residues in both the arnino and carboxy terminus of the protein
provide essentiai ligands for the trinuclear copper center (Zaitseva et al., 1996). Studies have
shown that ceruloplasmin plays a key role in the mobilization and oxidation of iron stores
and its subsequent incorporation into transferrin (Osaki and Johnson, 1969). This is
supported by the observation that animals deprived of dietary copper have little or no
circulating holoceruloplasmin and are unable to release iron into the plasma from tissue
stores (Roeser et al., 1970). Mutations in the ceruloplasmin geiie have been linked to
aceruloplasminemia, a disorder characterized by progressive neurodegeneration of the retinal
and basal ganglia (Harris et al., 1998). The mutations characterized so far have been s h o w
to abolish amino acids in the carboxy terminus which are critical for the formation of the
trinuclear copper cluster (Daimon et al., 1995; Hams et al., 1996; Hams et al., 1995;
Okamoto et al., 1996; Takahashi et al., 1996; Yoshida et al., 1995). This correlates with the
finding of little or no oxidase activity in the plasma of affected individuals (Harris et al.,
1998).
Lysyl oxidase is an extremely important enzyme, particularly during development,
since it is responsible for the formation and fùnction of comective tissue throughout the body
(Linder, 199 1). Lysyl oxidase is a secreted protein of about 32 kDa in size with one tightly
bound copper atom in the +2 oxidation state, ligated by four ligands (at least three of which
are nitrogen) in a distorted tetrahedral environment (Gacheru et al., 1990). The enzyme
functions by catalyzing the oxidative deamination of lysine and hydroxylysine side chains
and oxidizing the &-amino carbon to an aldehyde to form allysine- The resulting aldehyde
groups serve as the center for a cascade of spontaneous reactions involving the side chains of
non-oxidized lysine residues whicti results in the formation of inter- and intramolecular
crosslinks which stabilized the collagen and elastin fibers (Linder and Hazegh-Azam, 1996).
Like other copper oxidases, lysyl oxidase uses molecular oxygen and 6-hydroxy dopa to
cany out the oxidative deamination with the release of ammonia and peroxide.
Lysyl oxidase is widely expressed and is especially abundant in skin, veins, artenes
and other structures containing large amounts of comective tissue (eg. cartilage). It is often
isolated as four isoforms each with a molecular weight of 32 kDa. The enzymes are
synthesized as a pro-polypeptide of 50 kDa which is post-translationally modified by
proteolysis and glycosylatiou to produce the final mature form of 32 kDa (Romero-Chapman
et al., 199 1; Trackman er ai., 1992; Trackman et al., IWO). Little is known about how the
enzyme is regulated; however, many studies have pointed to copper as an important regulator
of Iysyl oxidase. Early reports on the effects of dietary copper deficiency on Iysyl oxidase
activity indicated that depressed copper levels resulted in decreased lysyl oxidase activity in
both chick aorta and lung (Harris, 1986; Harris et al., 1974). The lower lysyl oxidase levels
resulted in the proportion of salt extractable or non-crosslinked collagen to increase in these
tissues. Furthermore, the authors observed that feeding copper deficient animais trace
amounts of copper or adding it to cultures of aortic tissue resulted in the immediate
restoration of lysyl oxidase activity (Harris, 1976; Rayton and Hams, 1979). More recent
studies in rats have s h o w that even though copper deficiency results in a depression of lysyl
oxidase activity, the steady state levels of lysyl oxidase mRNA are unaffected (Rucker et al.,
1996). These results suggest that copper does not seem to bc involved in the regulation of
lysyl oxidase synthesis even though it is required for activity.
Superoxide dismutase (SOD) is a 32 kDa homodimeric protein which usually
contains one mole of copper and one mole of zinc per monomer. Manganese and iron
variants of the enzyme also occur, especially in bacteria (Linder and Hazegh-Azam, 1996).
The copper binding site in copper-zinc SOD is type 2, and the copper atom is coordinated by
four histidine residues, one of which serves as a bridging ligand between the copper atom and
the zinc atom. The copper and zinc sites are located at the bottom of a long narrow charnel
where the substrate diffùses into the active site. Mutation of one of the histidine residues
s~rrounding the copper to a cysteine results in the creation of a stable copper site closely
resembling that of type 1 copper (Lu et al., 1992). Although the activity of SOD requires the
presence of two essential metals, copper and zinc, its expression in many tissues is only
regulated by copper (Harris, 1992a; Harris, 1992). Copper-zinc SOD is present in the
cytoplasm of eukaryotic ceIls where it is responsible for the dismutation of superoxide (Oz*-)
to hydrogen peroxide (H202) and O2 through the cyclic reduction and oxidation of the bound
copper atom (McCord and Fridovich, 1969). Biochemlcal and immunological studies have
indicated that the enzyme is located in peroxisomes (Dhaunsi et al., 1992; Keller et al.,
1991).
It has also been shown that SOD is a critical enzyme in antioxidant defense. Oxygen
exposure has been shown to increase SOD activity as well as mRNA levels in both human
endothelial cells and yeast (Bermingham-McDonogh et al., 1988; Linder, 199 1). This
correlates well with the finding that transgenic mice with elevated SOD activity have
increased resistance to reperfusion injury after stroke, which in many cases is ascribed to
peroxidative processes (Maitre et al., 1993). Genetic analysis has shown that 10-15% of
familial amyotrophic lateral sclerosis ( F U S ) cases are linked to mutations in the SODl gene
(Deng et al., 1993; Rosen et al., 1993).
Many of the point mutations in the SODl gene Iead to a mutant protein which is still
able to bind copper and has normal superoxide scavenging activity (Corson et al., 1998).
These results suggest that loss of superoxide scavenging activity is not the primary cause of
FALS, indicating that FALS may be a result of a gain-of-fûnction associated with these
mutations (Gurney et al., 1994; Ho et al., 1998; Reaurne et al., 1996; Ripps et al., 1995;
Wong et al., 1995). The most promising theory puts forth the idea that these mutations lead
to a less restrictive active site, allowing access of other substrates which could lead to
aberrant chemistry at the copper atom. These substrates may include hydrogen peroxide
( H 2 0 1 ) and peroxynitrite (ON003 which have been shown to react with copper-zinc SOD in
virro to oxidize substrates and catalyze the nitration of phenolic groups, respectively
(Beckman et al., 1993; Hodgson and Fridovich, 1975; Ischiropoulos et al., 1992; Wiedau-
Pazos et al., 1996). The toxic products produced by these aberrant reactions may be the
cause of FALS; however, further work is required to c o n f m these hypotheses.
1.3 Copper Homeostasis in Humans
To avoid the toxic effects ofcopper excess or deficiency, an efficient method for the
uptake, distribution and excretion of copper is needed. Although many aspects of copper
transport have been studied for over the past 30 years, until recently little was known about
the molecular aspects of copper trûfficking within the body. The recent discovery of the
Wilson and Menkes copper transporting ATPases, together with the identification of copper
chaperones, has given us an unprecrdentltd understanding of the mechanisms behind copper
homeostasis. These discoveries and those to follow will be critical in developing new
treatments for disorders of metal metabolism.
1.3.1 Copper Lrptake and Circulation
Copper uptake in humans is confined mainly to the stomach and srnaIl intestines.
Although the overall mechanism of copper uptake in the intestines is not understood, several
studies have identified several key features of the system. It has been demonstrated in
several studies that copper uptake in the intestines is inversely related to the amount of
copper ingested (Tumlund et al., 1989; Tumlund et al., 1985). Thus with very high copper
diets, the absorption of copper in the intestines can be as low as 12% of maximum- A
theoretical absorptive maximum of 65% of ingested copper has been suggested, but, with
typical diets the actual copper uptake is about 40% (Wapnir, 1998). These observations have
strengthened the view that intestinal copper uptake plays a central role in the regulation of
overall copper absorption. Intestinal copper absorption is influenced by many dietary factors
such as pH, vegetable fibers, carbohydrates, and fats (table 1.4). The effect of pH on copper
absorption has been studied in the rat and it was found that the rate of copper absorption in
the ileum is higher at pH 5.8 or 7.8 thao at pH 6.3 - 7.3 (Wapnir and Stiel, 1987).
Sodium has also been suggested to be involved in the uptake of copper by the
intestinal mucosa. Perfusion studies using rat ileum and jejunum found that the rate of
copper uptake was significantly decreased when sodium was omitted fiom the perfusate
Table 1.4 Dietary Modifiers of Intestinal Copper Uptake in Humans
Modifier Effect Fibre
Hemicellulose &
Carbohydrates Fructose Glucose polymers
Fats Long-chain fatty acids Medium-chain fatty acids
Protein High-protein diet Excess amino acids
Organic acids Ascorbic acid Polybasic amino acids
Divalent cations (Zinc, Iron, Tin, Molybdenum) &
NK = Not Known
(Wapnir and Stiel, 1987). A similar result was also observed when amiloride was used to
inhibit the function of the sodium-proton antiporter and the sodium-calcium exchanger
(Wapnir, 1991). Although these results suggest a link between intestinal copper uptake and
sodium, the nutritional and biological significance of such a link is unknown. The effect of
carbohydrates, especially hc tose , has been studied extensively in rats. The effect of high
fructose diets in rats can lead to copper uptake insufficiency and these effects seem to be both
species- and sex-dependent (Fields et al., 1984; Reiser er al., 1983). This effect has also
been o b s e ~ e d in humans where high hc tose diets can Iead to decreased copper status
(Reiser et al., 1985). The widespread use of fructose-containing sweeteners and the finding
that copper sufficiency is closely linked to cholesterol metabolism makes these studies
especially relevant (Kievay, 1987).
The effect of amino acids on intestinal copper absorption is varied and is directly
linked to their concentration in relation to copper. One study in humans found that subjects
whose diet was supplemented with methionine had a net copper absorption which was nearly
double that of unsupplemented controls (Kies et al., 1989). Conversely, an excess of dietary
amino acids such as histidine and cysteine can have serious consequences on copper uptake.
In perhsion studies using rats, excess dietary histidine has been shown to cause increasing
losses of copper through the urine and to reduce copper uptake significantly (Harvey et al.,
1981). These observations probably stem from the ability of histidine to chelate copper,
thereby reducing its bioavailability, or competing for binding with the intestinal transport
proteins. A similar effect bas also been observed for cysteine. In addition to being an
effective chelator for copper, cysteine is also able to reduce copper from the divalent to
monvalent state, having a deleterious effect on copper uptake (Baker and Czarnecki-
Maulden, 1987). Other divalent cation such as zinc, ferrous iron, and starnous tin c m act in
a cornpetitive marner to decrease intestinal copper uptake (Hall et al., 1979; Pekelharing et
al., 1994; Van Campen and Scaife, 1967; Yu et al., 1994a).
The transport of copper from the intestinal lumen to the intestinal mucosa is a canier-
mediated process involving at least one saturable component (Crampton et al., 1965;
Tumlund et al., 1989). Although the identity of the intestinal copper transport protein is not
known, the Menkes disease copper transporting ATPase is a strong candidate in light of the
Menkes phenotype and the strong expression of the protein in the intestinal mucosa (Chelly
el al., 1993; Mercer et al., 1993; Vulpe et al., 1993). Once copper enters the blood Stream it
becomes bound to a variety of proteins. More than 90% of copper is bound to ceniloplasmin,
a large multicopper senun glycoprotein; however, this copper is tightly bound and considered
non-exchangeable (Linder and Hazegh-Azam, 1996). The remaining 5 - 10% is
exchangeable and is bound to albumin, and amino acids as iow molecular weight complexes.
Several carefiil and detailed studies revealed that the main copper-amino acid complex in the
serum was in the form of a 1 :2 copper-histidine complex (Neumann and Sass-Kortsak, 1967;
Sarkar and Kruck, 1966). Further studies also revealed that histidine could form a temary
complex with albumin and copper (Lau and Sarkar, 1971 ; Sarkar and Wigfield, 1968). The
copper binding site on albumin has been very well characterized (Iyer et al., 1978; Laussac
and Sarkar, 1980; Laussac and Sarkar, 1984). The copper binding site on albumin is at the
amino terminus and involves the first three residues (Asp-Ala-His). Copper is ligated in a
pentacoordinate arrangement using the amino-terminal nitrogen, the carboxyl side chain of
aspartic acid, two intervening peptide nitrogens, and an imidazole nitrogen fiom a histidine
residue in the third position. The four nitrogen ligands are in a square-planar arrangement
with the carboxyl oxygen occupying the axial position.
Copper bas also been observed to bind to another large semm protein found in rats
known as transcuprein (Linder, 199 1; Weiss and Linder, 1985). Since this protein has yet to
be cloned and has not been fully characterized, conclusions cannot be drawn regarding its
biological function (Barrow and T a ~ e r , 1988; Goode et al., 1989; Tsai et al., 1992). It is
clear that albumin is responsible for carrying the majority of newly absorbed copper ions in
the blood stream. Approximately 40 pg of copper are requircd to fil1 al1 the high affinity
albumin copper sites in I ml of human serum, but the amount that actually binds in vivo is far
less (Linder and Hazegh-Azam, 1996). Although albumin is an important transporter of
copper in the blood stream, its role in the uptake of copper by various tissues remains
unknown. When albumin is completely absent, as is the case in analbuminernia, there does
not seem to be any disruption in the distribution of copper (Vargas et al., 1994). Although
albumin levels in the nagase rat are 4000 times lower than normal, intravenously injected
67 Cu entered the liver at least as fast in these animals as in normal animals and its
reappearance in ceruloplasmin seemed to be accelerated. These findings suggest that
aIburnin may be playing a passive role in copper transport, acting as a reservoir for newly
absorbed copper. This view is strengthened by the observation that copper uptake by
hepatocytes is inhibited by albumin and no ce11 surface receptors for albumin have been
found on these cells (Ettinger et of., 1986). It is likely that albumin-bound copper must
exchange with other components, which then facilitate its uptake into cells. Transfer of
copper between albumin and transcuprein has been shown, as has exchange between albumin
and histidine (Lau and Sarkar, 1971; Sarkar and Wigfield, 1968; Tsai et al., 1992; Weiss and
Linder, 1985). The former may be important in rodents while the latter is more important in
humans.
1.3.2 Membrane Transport of Copper
There are at least two pathways by which cells take up copper from the bloodstream
(fig. 1.1). The first pathway involves copper release and uptake from ceruloplasmin.
Ceruloplasmin has been shown to be able to donate copper to cells as well as to intracellular,
copper-containing proteins (Campbell et al., 198 1 ; Dameron and Hams, 1987; Hsieh and
Frieden, 1975; Marceau and Aspin, 1973). The uptake of copper from ceruloplasmin is
belicved to occur via interaction witb a ce11 surface ceruloplasmin receptor, releasing
ceruloplasmin bound copper. The released copper is then reduced by a ce11 surface reductase
and enters the ce11 through an energy-independent transport protein. Although the nature of
the uptake protein has not been established conclusively, hCTRl has been proposed to be the
high affinity copper uptake protein in humans (Zhou and Gitschier, 1997). This protein was
initially discovered by its ability to complement the yeast hi@ affinity copper uptake mutant,
ctrl. The human CTRl protein shares 29% identity with its yeast couterpart; however, it is
much smaller in both the intracellular and extracellular metal binding domains (Zhou and
Gitschier, 1997). A second protein, hCTR2, which is similar to hCTRl was discovered
through database searching and both genes are expressed in al1 tissues. The exact role of
these proteins in the uptake of copper remains to be established.
l
[ Intestine
Figure 1.1 Intestinal and Cellular Copper Upiake Pathways. Copper is absorbed from the gastrointestinal tract and enters the exchangeable pool. During uptake, copper is reduced to Cu(1) by a hypothetical membrane reductase and is absorbed by the ce11 through a passive transporter, possibly hCTRI. In the ceruloplasmin (Cp) pathway, copper is released Erom Cp through interaction with its receptor, followed by reduction and uptake. Once inside the cell, copper becomes bound to glutathione (GSH) and various copper chaperones (ATOXl , Ccs 1, Coxl7). The Wilson disease ATPase is positioned in the bans-golgi membrane where it pumps copper into the excretory pathway for incorporation into cuproproteins.
htative ceruloplasmin receptors have been identified in a variety of tissues and ce11
types; however, those which have been isolated, differ in size and composition (Barnes and
Frieden, 1984; Gordon et al., 1987; Kataoka and Tavassoli, 1 984; Kataoka and Tavassoli,
1985; Omoto and Tavassoli, 1990; Orena er al., 1986; Stem and Fneden, 1993; Stevens et
al., 1984). These differing proteins may represent different components of the ceruloplasrnin
receptor, al1 of which have some affinity for ceruloplasmin. Studies on the uptake of
ceruloplasmin-bound copper by human erythroleukemia celIs revealed that ceruloplasmin
itself was not intemalized when copper was taken up by the ce11 (Percival and Harris, 1990).
This study also found that the addition of ascorbate strongly stimulated uptake of copper
while the addition of cuprous chelators inhibited it. In addition, sulfhydryl-modifying
reagents were also found to inhibit uptake, implicating cysteine residues in the copper upbke
process. Taken together these results suggest that copper is taken up in the monovalent
rather than the divalent form (Harris, 199 1; Percival and Harris, 1989).
The second pathway involves uptake f?om non-ceruloplasmin-bound copper such as
that bound to histidine, albumin, and other low molecular weight complexes (Hams and
Sass-Kortsak, 1967; Sass-Kortsak et al., 1967). As is the case for the ceruloplasmin
pathway, uptake of copper does not involve intemalization o f its ligands (Ettinger et al.,
1986; van den Berg and van den Hamer, 1984). Studies on the kinetics of copper uptake by
cultured hepatocytes support a system which is carrier-mediated, energy-independent and
saturable (Ettinger et al., 1986; McArdle et al., 1988; Schmitt et al., 1983; Stockert et al.,
1986; Weiner and Cousins, 1980). Uptake via this pathway can also be inhibited by other
metals such as zinc or cadmium, reflecting the similar coordination preferences of these
rnetals, or suggesting that the protein involved is a general metal carrier (Ettinger et al., 1986;
Schmitt et al., 1983). As has been mentioned previously, histidine and albumin have
different effects on the uptake of copper. Ln the absence of albumin, at ratios less than 1 : 1
witb copper, histidine has no effect on copper uptake. At higher concentrations, histidine
acts as a weak cornpetitive inhibitor of copper uptake in hepatocytes, but, in the presence of
albumin, stimulates the uptake of copper (Dmvish et al., 1984). Studies on the uptake of
copper by placenta1 cells shows that histidine stimulates copper uptake in the presence of
serum while albumin inbibits it (Mas and Sarkar, 1992). These findings and the observation
that analbuminemia in humans and animals does not result in a disturbance of copper uptake
strongly suggest that histidine plays a central role in the uptake of copper from the
bloodstream.
1.3.3 Intracellular Copper Tra ffrcking
Once copper is taken up by the cell, it enters a series of complex pathways which are
only now becoming understood (fig 1.2). Much of the absorbed copper is immediately
excreted from the ce11 via pathways which wil1 be discussed in the following section. Botb
copper and copper proteins are distributed throughout the ce11 and are present in al1 cellular
organelles including lysosomes, mitochondria, endoplasmic reticulum, nucleus and the
cytosol (Vulpe and Packman, 1995). Examples include metallothionein (MT) in the nucleus,
cytosol, and lysosomes (Janssens et al., l984a; Janssens et al., 1 984), cytochrome c oxidase
in the rnitochondria (Kodama et al., 1989), Iysyl oxidase in the golgi and secretory organelles
(Kuivaniemi et al., 1986), and superoxide dismutase (SOD) in the cytosol and possibly
peroxisomes (Crapo et al., 1992). For many years the mechanism by which copper was
delivered to various proteins and cellular compartments was not understood. Recently a
family of small cytosolic proteins has been identified which are responsible for the delivery
of copper to specific cellular targets. These proteins have corne to be known as copper
chaperones. Al1 of these proteins were initially identified in yeast and their human
orthologues were identified by complementation studies.
The copper chaperone family currently includes three members: ATXlhuman
ATOX 1 (formerly known as HAH1) (Hung er al., 1998; Klomp et al., 1997; Lin et al.,
1997), Lys7/human Ccsl (Culotta et al., 1997; Gamonet and Lauquin, 1998), and
Cox 17/human Cox 17 (Amaravadi et al., 1997; Beers et al., 1997; Glerum et al., 1996). Each
of these proteins delivers copper to a different copper containing protein. ATXl delivers
copper to CCC2 (the yeast homologue of WND), lys7 delivers copper to superoxide
dismutase and Coxl7 delivers copper to the mitochondria for incorporation into cytochrome
c oxidase. A direct interaction between these chaperones and their target proteins has only
been demonstrated in two cases. ATXl and the amino-terminal domain of CCC2 have been
shown to interact using yeast two-hybrid analysis (Pu fahl et al., 1997). In the case of Ccs 1
an in vitro interaction with SOD was demonstrated using fusion proteins and an in vivo
interaction was demonstrated by CO-irnmunoprecipitation (Casareno et al., 1998). Although
it is known that Cox17 is responsible for delivering copper to the mitochondria, no specific
target has yet been identified. A possible target of Coxl7 that has been proposed is Sc01
(Glerum et al., 1996). Sc01 is a mitocbondrial protein which has been shown to be critical
for the proper assembly of cytochrome c oxidase and has been localized to the inner
mitochondnal membrane (Buchwald et al., 199 1 ; Krurnmeck and Rodel, 1990; Petnizzella et
al., 1 998; Schulze and Rodel, 1 988). Despite these suggestive results, a direct interaction
between Coxl7 and Sco 1 has not been demonstrated in vivo or in vitro
In most cases the structure of the chaperone is very similar to its target. The primary
structure of Atxl is very similar to the amino-terminal metal binding repeats in CCC2
(Pufahl et al., 1997). Atxl also contains one copy of the metal binding motif, Met-Thr-Cys-
Xaa-Xaa-Cys, which is most likely involved in metal ligation and is present in two copies at
the amino terminus of CCC2 (Yuan et al., 19951. Similarly, the primary structure of Ccsl
bears a striking similarity to the carboxy-terminus of SOD. Siace SOD is known to
homodimerize, this similarity has Iead to the idea that the chaperone dimerizes with SOD to
facilitate the transfer of copper between the two proteins.(Casareno et al., 1998; Culotta et
al., 1997). Since the cellular target of Coxl7 is unknown, no cornparisons can be made
regarding similarity of structure. The copper binding site of Coxl7 has been characterized
spectroscopically. The protein was found to contain two copper atoms in a binuclear
cuprous-thiolate complex which is substantially more labile than similar clusters in other
proteins such as metallothionein (Srhivasan er al., 1998).
Glutathione is also another major chelator of copper in the cytoplasm; however, its
function may be more important in the case of copper overload than under normal conditions.
Studies on copper-loaded hepatoma cells have indicated that over 60% of cytosolic copper
exists as the copper-glutathione complex (Freedman et a/., 1989). The copper-glutathione
complex can also donate copper to various proteins. In viro studies have shown that the
copper-glutathione complex can donate copper to apo-SOD, apo-metallothionein, and apo-
ceruloplasmin (Cino10 et al., 1990; Ferreira er al., 1993; Musci et al., 1996).
Metallothioneins are small molecular weight (6.5 D a ) metal binding proteins which
have several distinguishing features. They have a high metal binding capacity (7 - IO g
atoms/mol); 30% of the amino acids are cysteine yet the native protein has no disulfide
bonds, are devoid of aromatic residues, and exhibit and substantial amount of polyrnorphism
(Cousins, 1985). Metallothionein was originally isolated as a cadmium and zinc binding
protein, but is usually found naturally as a zinc binding protein (Kagi and Vallee, 1960).
Indeed many of the biological fùnctions of metallothionein seem to revolve around zinc
metabolisrn (Cousins, 1985). It has been suggested that metallotbioneins acts as a reservoir
for zinc, regulating its intracellular availability. In support of this notion, the reversible
exchange of zinc between rnetallothionein and the estrogen receptor (2 zinc finger protein)
has been demonstrated, as has the removal of zinc fiom zinc finger-containing transcription
factors such as Sp 1 and TFIILA (Cano-Gauci and Sarkar, 1996; Zeng et al., 199 1 a; Zeng et
al., 1991).
Metallothionein synthesis can be induced by a variety of factors including heavy
metals, physical stress, glucocorticoids, and heat shock. Metallothionein is able to bind other
heavy rnetals such as copper, cadmium, iron, and mercury (Kagi and Schaffer, 1988).
Although the level of metallothionein induction by zinc is greater than that by copper,
metallothionein has a higher binding affinity for copper than zinc (Lipsky and Gollan, 1987).
It is unlikely that rnetallothionein plays a central role in the intracelluIar trafficking of
copper, and its primary role in relation to copper may be one of detoxification and storage.
Many studies have shown that copper-metallothionein accumulates in the lysosomes of
copper-loaded mimals, probably as a protective rneasure of the hepatotoxic effects of excess
copper (Gooneratne et al., 1980; Johnson et al., 198 1 ; Verity et al., 1967). The copper stored
in metallothionein can be made available to other proteins either following metallothionein
degradation in lysosome (Mehra and Bremner, 1983) or by exchange via complexation with
glutathione (Freedman et al., 1989). Yeast strains which have had their rnetallothionein-like
gene knocked out are able to thrive on normal media and maintain normal levels of copper
enzymes, but are intolerant to copper stress. Expression of mammalian metallothionein in
these strains restores their ability to grow in high copper media, supporting the notion that
metallothioneins play a major role in copper detoxification and are not centrai to normal
intracellalar copper metabolism (Thiele et al., 1986). It is likely that a similar scenario takes
place in mammalian cells.
1.3.4 Excretion of Copper
The efficient excretion of excess copper is an integral component of proper copper
metabolism, and the liver plays an important part in this process. Upon being absorbed by the
liver, copper may be retumed to circulation by incorporation into ceruloplasmin,
incorporated into copper containing enzymes, stored within metallothionein, or excreted into
the bile. Urinary copper excretion is negligible under normal physiologic conditions since
most of the circulating copper is either bound to ceruloplasmin or confiaed within
erythrocytes, and little or no copper penneates the glomemlar capillaries (Owen Jr, 1964).
The smaIl amount of ingested copper that does appear in the urine most likely originates
frorn the amino acid-bound fraction of plasma copper and the dissociation of the copper-
albumin cornplex. Ln the overall scherne of copper homeostasis, urinary excretion does not
appear to play an important role. However, cupmria does occur in the later stages of
pathological conditions which affect primary copper homeostatic mec hanisms such as biliary
copper excretion, copper storage and cemloplasmin synthesis.
Biliary excretion is an important mechanism for the excretion of many trace metals
(table 1.5) and accounts for -80% of the copper leaving the liver (Evans, 1973). The
Table 1.5 Metal Content of Rat Bile
-
Metal Concentration (PM) Manganese 5.4 k 0.8 Iron Copper Zinc Molybdenum Vanadium Chromium Strontium Lead 2.8 = 0.7
Adapted fiom @ijkstra et ai., 1 996).
exact mechanism of copper excretion into the bile is not known; however, three potential
routes can be distinguished: i) lysosomal exocytosis, ii) a glutathione-dependent route (via
glutathione conjugate transporter), and iii) secretion by a copper transporting ATPase. The
idea that lysosomes are the source of copper which is excreted into the bile stems from a
large number of studies which have s h o w that large amounts of copper are stored within
hepatic lysosomes under certain physiologie conditions (Mohan et al., 1995; Sternlieb,
1989). In a kinetic study where 64copper was administered orally to a single Wilson disease
patient the specific activity of hepatic lysosomes was similar to that found in the bile
(Sternlieb et al., 1973). This finding, together with the observation that lysosomes could
normaIly release their contents into the bile by exocytosis (De Duve and Wattiaux, 1966;
LaRusso and Fowler, 1979), strengthened the idea that lysosomes are the primary source of
biliary copper. Studies in copper-loaded rats have also shown an association betweeo biliary
copper output and the activity of lysosomal enzymes in bile (Gross et al., 1989) and the
authors suggest that exocytosis of lysosomal contents into bile represents a major mechanisrn
for biliary copper excretion under these conditions. Much of the copper stored in lysosomes
has been found to exist as a complex with a polymerized form of metallothionein (Gross et
al., 1989).
Glutathione (GSH) has been implicated in the biliary excretion of copper since the
early eighties when it was shown that administration of diethylmaleate (DEM) to rats lead to
a strong reduction in the biliary excretion of copper, concorninent with a decrease in hepatic
GSH levels (Alexander and Aaseth, 1980). The inhibitory effect of DEM is also seen with
intravenously administered copper following DEM treatment. DEM is able to forrn
conjugates with GSH, leading to a lowering of available hepatic and biliary GSH. Analysis
of bile fluids fkom rats after copper administration revealed that copper in the bile is
conjugated to GSH (Alexander and Aaseth, 1980). In studies with normal rats it was noticed
that intravenously administered copper was eliminated in a biphasic manner composed of a
rapid phase immediately after injection, followed by a slow gradua1 phase (Nederbragt,
1989). in normal rats the rapid phase is inhibitable with DEM, but the slow phase is not,
suggesting different mechanisms for each process.(Houwen et al., 1990). This has been
confirmed in the mutant GY rat which, despite doubled hepatic GSH levets, is unable to
secrete GSH into the bile. In these animals, the rapid phase is absent; however, the slow
phase as well as secretion of endogenous copper do not seem to be affected (Dijkstra et al.,
1990; Houwen et al., 1990).
These studies strongly suggest that an adequate supply of intracellular GSH is
required for the proper excretion of both endogenous copper and intravcnously administered
copper into bile. However, the rapid phase of biliary copper excretion is only present when
GSH is able to be secreted into the bile. In light of these findings, it seems probable that the
rapid phase involves transport of copper-GSH complexes into bile (fig. 1.2) and this would
most likely occur through the canalicular multispecific organic anion transporter (cMOAT)
(Lee et al., 1997). This finding has been fùrther elaborated by studies of dietary copper
overload in mutant GY rats. In these studies, it was shown that GY rats fed hi@ copper diets
accumulated copper in their livers to the same extent as control animals and had the same
rate of biiiary copper excretion (Dijkstra et al., 1990; Houwen et al., 1990). This implies that
GSH and cMOAT are not involved in a i s process, leading the authors to suggest that there is
another saturable copper transport system in the rat liver.
The Wilson disease copper transporthg ATPase (WND) has been implicated as this
saturable component in the rat Iiver. Although some have demonstrated ATP dependent
transport in isolated rat liver plasma membranes (Dijkstra et al., 1999, localization studies
have not identified the presence of the Wilson disease ATPase on the canalicular membrane.
However, WND may still play an important part in biliary copper excretion. Localization
studies have shown that WND resides in the trans-golgi under steady-state conditions, but
undergoes a reversible, copper-specific translocation to a cytosolic vesicular cornpartment
under elevated copper concentrations (Hung er al., 1997). These vesicles could be
lysosomes; however, ATP dependent copper transport in pure lysosomal membranes has not
been evaluated. The ATPase would pump copper into this vesicle, which could then fuse
with the canalicular membrane and dump its contents into the bile. In this case, GSH has
been postulated to reduce copper(1I) to copper(I), leading to the formation of copper(1)-GSH,
which could donate copper(1) to WND. The copper chaperone, ATOXI, would also be
involved in the donation of copper to WND, in addition to the copper(1)-GSH pathway. This
step may be critical in the efficient transport of copper by this ATPase, as has been
demonstrated for bac terial copper transporting ATPases (Solioz and Odermatt, 1995). In
either case, it is clear that GSH plays a central role in the biliary excretion of copper.
1.4 Cation Transporting ATPases
Cation transporting ATPases (also known as ion motive ATPases) are responsible for
the translocation of ions across biological membranes and are critical for the maintenance of
ion gradients, proper intracellular ion composition and ion resistance. These membrane
bound purnps are found in bacteria, plants, hngi and animals and function by using the
energy obtained Erom the hydrolysis of ATP to move the ion across the membrane. The
known ATPases have been classified into three major groups; P-type, V-type, and F-type
(FIFo-type) ATPases. P-type ATPases are distinguished fiom the other two types by the
formation of a phosphorylated intennediate during the transport cycle. V- and F-type
ATPases are stnicturally and genetically related; however, they have different mechanisms of
action. F-type ATPases are normally run in reverse using an ion gradient to generate ATP.
An example of this is the ATP synthase, Iocated in the imer mitochondrial membrane, which
uses the H- gradient generated from oxidative respiration to drive the synthesis of ATP fiom
ADP (Fillingame, 1997). In contrast to FrFo ATPases, V-type or vacuolar ATPases function
exclusiveiy as proton pumps using the hydrolysis of ATP to drive the reaction. V-type
ATPases are involved in energizing intracellular compartments of the vacuolar system
providing an electrochemical gradient for the transport of solutes and acidifLing several of
these compartrnents (Gluck, 1992).
Recently, two genes which are defective in two human genetic disorders of copper
metabolism (Menkes and Wilson disesise) have been s h o w to encode copper transporting P-
type ATPases (MNK and WND respectively). These were the first heavy metal transporting
ATPases identified in humans. They are part of a larger family of heavy metal transporters
found in bacteria and yeast, responsible for the transport of a diverse set of metals, including
copper, cadmium, zinc, mercury and arsenic (Siiver, 1996). Although similar in many
respects to other P-type ATPases, the heavy metal ATPases have some unique features, the
hnctions of which are beginning to be elucidated. The following sections will provide a
brief outline of the structural features of each type of ATPase noting similarities and
di fferences.
1.4.1 Types of Cation Transporting ATPases
1.4.1.1 P-TypeATPmes
Characteristic features of P-type ATPases which distinguish h e m fiom the other two
type (V and F) are the formation of a phosphoenzyme intermediate, cycling between two
distinct forms (El and Eî) and inhibition by vanadate (Moller et al., 1996). The mechanisrn
of action of two extensively studied P-type ATPases the Na-IK--ATPase (Glynn and Karlish,
1990) and the sarco(endo)plasmic reticulum c ~ ' - - A T P ~ s ~ (SERCA) (MacLennan et al..
1997a) are depicted in figure 1.2. Generally, this mechanism can be represented by a four
step reaction El -. EIP -. EzP -. Ez -. El and is referred to as the Post-Albers scheme (Post
et al., 1972). in the first step, a phosphorylated intermediate (ElP), with bound cations, is
forrned by the reaction of the enzyme with MgATP. This is a high energy intermediate
which can be dephosphorylated with ADP reforming ATP. Following phosphorylation of the
protein, structural changes occur which lead to the translocation of the bound ions across the
membrane. This in turn resuhs in the conversion of the phosphorylated enzyme to the low-
energy E2P form which is unreactive with ADP. In this state, the afinity of the enzyme for
its respective ions is low. The final steps in the reaction invoive removal of the phosphate
group by hydrolysis and reformation of the El state. in many cases reforming the El state
involves the transfer of other ions, K' for the Na'/K'-ATPase and H for SERCA (Levy et
al., 1990; Yu et ai., 1993). The vanadate ion ( ~ 0 4 ~ 3 is a potent inhibitor of P-type ATPases.
It is a transition state analogue of phosphoryl transfer reactions because it readily adopts a
pentacovalent bipyramidal geomeûy. Its inhibitory action most likely stems from its ability
to form a stable transition state complex with the ATPase (Cantley et al., 1978).
a
1 ATP ADP
2ca2+ - fi-
Figure 1.2 Transport Mechanisrn SERCA and N~'/K' ATPases. A SERCA mechanism. Calcium and ATP bind in the cytoplasm resulting in phosphorylation of the El enzyme (1) which undergoes a conformational change to the EL state (2) releasing calcium into the lumen (3), the enzyme is then dephosphorylated and the El state is reformed (4). B ~a' /J f mechanism. Sodium and ATP bind in the cytoplasm resulting in phosphorylation of the EI enzyme (1) which rapidly changes conformation to E2 (2). A series of reactions then occur, which rcsults in the release of sodium and binding of potassium outside the cell, followed by dephosphorylation of the EI enzyme (3). The dephosphorylated El enzyme undergoes another conformational change back to El, with the release of potassium into the cytoplasm.
The P-type ATPases can be divided into two main groups based on the type of ion
transported, either heavy-metal (Type I) or non-heavy-metal (Type Il) (figure L -3). Ali heavy
metal transporting ATPases have similar hydropathy profiles which are significantly different
from those of non-heavy metal transporting ATPases. Members of the type 1 group include
bacterial copper and cadmium ATPases (Nucifora et al., 1989; Odennatt et al., 1993), the
nitrogen fixation protein fixI (Kahn et ai., 1989), the human copper transporting ATPases
(MNK and WND) (Bull er al., 1993; Tanzi et al., 1993; Vulpe et al., 1993) and rnany others.
Al1 members of this family have between 6 and 8 transmembrane segments with only one
pair of transmembrane segments on the carboxy-terminal side o f the cytoplasmic ATP
binding domain. They also contain a highly hydrophobie region, immediately preceding the
TGES/A conserved motif, which probably gives rise to two additional transmembrane
segments in this region. This is in contrast to members of the type II family which contain
only one pair of transmembrane segments in this region (Lutsenko and Kaplan, 1995).
However, the most striking and interesting feature of the type 1 ATPases is the presence of a
large amino-terminal cytosolic domain which contains between one and six copies of the
metal binding repeat GMTCXXC or M/HXXMDHS/GXM (Bull and Cox, 1994; Petnikhin et
al., 1994; Silver et al., 1993). The variable number of these repeats leads to a great diversity
of molecular weights among members of the type 1 family of ATPases.
In contrast to type 1 ATPases, the type II, non-heavy metal transporting ATPases only
have one pair of transmernbrane segments before the TGES energy transduction motif.
Members of this family have a catalytic subunit which consists of 8 to 10 transmembrane
segments and are approximately 100 kDa in size (Lutsenko and Kaplan, 1995). The type II
ATPases can be further subdivided into two groups IIA and IIB. Type IIA
Figure 1.3 Topology of Type I und Type II P-Type ATPuses. Type 1 (heavy metal) and Type II (non-heavy metal) P- type ATPases. White blocks represent core structure, gray blocks represent putative or additional trammembrane segments. Cooserved motifs are indicated, TGES (energy tranduction), DKTG (phosphorylation site), GDGXDN (hinge reg ion), SEHPL (unknown fÙnc tion, type 1 only), GMTCXXC or M/HXXMDHS/GXM (metal binding, n = 1-6).
ATPases have a heterodimeric structure consisting of a catalytic a-subunit and a
glycosylated f3-subunit. Examples of type IIA ATPases include the Na-/K- and H-/K-
ATPases which exchange intracehlar Na' or HT for extracellular K- (Glynn and Karlish,
1990; Sachs er al., 1989). Type IIB ATPases fimction as monosubunit proteins transporting
divalent cations into intracellular compartments or into rhe extracellular milieu. Members of
this family include the calcium ATPases of the plasma membrane and sarco(endo)plasmic
reticulum and the H*-ATPases of fûngi and plants (Moller et al., 1996). The bacterial
inducible, high affinity K' uptake ATPase (KdpK*-ATPase) is somewhat diffèrent kom both
type 1 and type II ATPases. Like the type II ATPases, this protein has only one pair of
transmembrane segments before the TGES motif and bas no metal binding sites at the amino-
terminus. However, it is also missing two transmembrane segments at the carboxy-terminus
which make it shorter than other type II proteins and, in this respect, it resembles the type 1
proteins. Unlike the heavy metal transporters which fiinction as monomers (Nucifora er al.,
1989; Tsai and Linet, 1993), the KdpK--ATPase is active as a multisubunit complex. The
complex consists of three subunits, A, B, and C, which contain 557, 682, and 189 amino
acids, respectively (Hesse et al., 1984). The phosphorylation site is locatsd in subunit B,
while subunits A and C corne together to form the transmembrane charnel (Altendorf et al.,
1 994; Buurrnan et al., 1995)
The core structure of P-type ATPases contain many conserved motifs. Among these
are the TGES, DKTG, and GDGXDN motifs. The TGES motif is located in the cytoplasmic
Ioop between transmembrane segments 2 and 3, which is sometimes referred to as region B
(Moller et al., 1996). This region bas been implicated in energy transduction based on site
directed mutagenesis of the TGES residues and proteolytic cleavage experiments at residues
close to this motif in the SERCA ATPase (Clarke et al., 1990; Imamura and Kawakita, 1989;
Juul et al., 1995; le Maire et al., 1990). These mutagenesis and cleavage experiments
resulted in proteins which retained caIcium binding and phosphorylation capacity, but were
not able to transport calcium. Based on these results, it has been hypothesized that
modifications in this region prevent the conformational change fiom the EiP fonn to the E-P
forrn, leading to an accumulation of EiP with bound calcium (Moller et al., 1996).
The DKTG motif is located in the large cytoplasmic loop between transmembrane
segments 4 and 5 and is referred to as domain C. The DKTG motif is critical to the function
of the ATPase, since it contains the conserved aspartate residue which is phosphorylated in
the transport cycle of the protein. Mutation analysis in the SERCA ATPase has shown that
both the aspartate and neighboring lysine residue are needed for phosphorylation to take
place and cannot be replaced by any other residues (Maruyama and MacLennan, 1988).
Furthemore, mutation of these residues or transposition of the aspartate and lysine rssidues
(DK -. KD) results in loss of phosphorylation and calcium transport (Maruyama and
MacLennan, 1988). These results suggest that the lysine residue may be part of a
stereospecific, acid-base catalyzed transfer of phosphate fiom ATP to the aspartate residue.
The GDGXDN 'hinge' motif is located at the end of domain C just before it enters the
membrane again with trammembrane segment 5 and is referred to as rcgion J. This region is
predicted to be highly alpha-helical and has been predicted to form part of the 'stalk' of the
ATPase (Moller et al., 1996).
The hinge motif also contains two aspartate residues which seem to be required for
phosphorylation, as indicated by chemical modification and mutagenesis studies on both the
N~*/K+ and SERCA ATPases (Lane et al., 1993; Ohta et al., 1986; Vilsen er al., 199 1).
Modification studies in the N a X ' ATPase also indicate that this region may hteract closely
with the ATP binding region of the protein (Ohta et ai., 1986). The SHEPL motif is only
present in heavy metal transporting P-type ATPases in dornain C, just after the
phosphorylation site. The histidine in this motif is cntical for proper h c t i o n of the protein
since mutation of this residue to serine in the Wilson disease copper ATPase leads to the
Wilson disease phenotype (Petrukhin et al., 1993). Although no specific function has yet
been ascribed to this motif in the metal ATPases, the analogous region in SERCA and plasma
membrane Ca-ATPases is a site of regulation through interaction with phophotamban (James
el al., 1989; Toyofiiku et al., 1993) and calmodulin (Falchetto et al., 199 1 ) respectively.
1.4.1.2 Y-gpe and F-type A TPases
V-type (or vacuolar) ATPases are stnicturally related to F-type ATPases and hence
will be considered together in this section. V- and F-type IT ATPases differ £iom the P-type
ATPases in two important ways. First, V- and F-type ATPases are large multisubunit
complexes, in contrast to P-type ATPases which usually function as monosubunit proteins.
Second, although they utilize the energy of ATP hydrolysis, V- and F-type ATPase do not
utilize a phosphoenzyme intermediate (Nelson and Taiz, 1989). V-type ATPases pump
protons from the cytoplasm to the lumen of vacuoles and other cellular organelles such as
lysosomes, endoplasmic reticulum, golgi bodies and secretory granules. As such, they are
closely involved in the maintenance of cellular pH and energizing intracellular compartments
(Finbow and Harrison, 1997). Since the V-type ATPase does not use a counterion, its
activity is electrogenic, creating an electrical potential difference (AY) across the membrane.
This membrane potential is then used to drive solutes and other ions across the organelle
membrane, as occurs in the accumulation of neurotransmitters in synaptic vesicles and amino
acids in the vacuole lumen (Nelson, 1994). The acid pH conditions created by the pumping
of protons into vacuoles is used for othet functions as well (Mellman, 1992). In lysosomes,
low pH is needed to activate acid hydrolases and the dissociation of receptor-ligand
complexes (eg. Fe(iI1) fiom transferrin) (Brown et al., 1983). V-type ATPases are also
closely involved in the trafficking within the endosomal / lysosomal compartment.
Bafiiomycin Al, a specific inhibitor of V-type ATPases, causes a decrease in the rate of
receptor cycling (Johnson et al., 1993). It has since been found that acidification is a
necessary step in the formation of endosome camer vesicles, which are intermediates
between early and late endosornes (Clague et al., 1994).
F-type ATPases (also known as FIFo-ATP synthases) fùnction in reverse when
compared to V-type ATPases. Whereas V-type ATPases use the hydrolysis of ATP to drive
the creation of a membrane potential, F-type ATPases use a membrane potential to dnve the
synthesis of ATP from ADP. In animal cells, the enzyme is located in the inner
mitochondriai membrane where it uses the proton gradient established by oxidative
respiration to drive the synthesis of ATP. Closely related F-type ATPases are found in the
thylakoid membrane of plant chloroplasts, and in the plasma membrane of bacteria such as E.
coli. In plants, the enzyme functions in electron transport-driven photophosphorylation,
while in bacteria the pump can operate reversibly depending on the nutritional status of the
ce11 (Fillingarne, 1997). Under aerobic growth conditions, a proton gradient is created across
the plasma membrane by electron transport systems by pumping protons from the cytoplasm
to the outside of cell. The proton gradient is then used to generate ATP and is also utilized in
proton-coupled nutrient transport and flagellar rotation (Fillingame, 1997). Under anaerobic
conditions, the proton gradient is generated by the hydrolysis of glycolytically derived ATP
by the FiFo-ATPase, pumphg protons fÎom the cytoplasm to the outside of the cell. Al1 three
FI Fo-ATPases (mitochondrial, chloroplast, and bacterial) are stnicturally and mechanistically
related, however they are regulated differently (Harris, 1995; Mills et al., 1995; Walker,
1994).
The F-type ATPases are composed of two structurally and functionally distinct
sectors terrned FI and Fo. The catalytic sector FI extends Erom the surface of the membrane
as a knob-like projection. The Fi sector c m be removed from the membrane as a water
soIuble protein capable of hydrolyzing ATP (Fillingame, 1997). The F i sector is connected
to the membrane bound Fo sector via a narrow stalk. Wben the Fi sector is not present, the Fo
sector can mediate the passive transport of protons across the membrane. Once properly
assembled, the complex mediates the reversible coupling of ATP hydrolysis to proton
transport. The bacterial FIFo-ATPases are cornposed of eight types of polypeptides in the
following stoichiometry: a 3 B 3 y t k (FL) and ai6,c12 (Fo) (Fillingame, 1997). The a3P3y portion
of FI from beef heart mitochondria has been crystallized and structural analysis shows a
symmetncal hexameric arrangement of alternating a and f3 subunits (55 kDa each) around a
central core (Abrahams et al., 1994). The ATP binding sites are Iocated within the subunit
at the af3 subunit interface. The y subunit (30 kDa) is located in the center of the hexamer
and protrudes partially fiom the bottom of the complex. The three dimensional arrangement
of the subunits in the FO domain is unknown.
The V-type ATPases, like their F-type counterparts, are cornposed of a catalytic VI
sector and a membrane bound Vo sector. The VI sector c m be removed using a variety of
methods, including chaotropic agents, alkaline carbonate, or nitrate and MgATP (Kane et al.,
1989; Lai et al., 1988; Xie and Stone, 1988). Unlike the Fo sector, there is no evidence to
suggest that the Vo sector can passively transport protons in the absence of VI (Finbow and
Harrison, 1 997). The subunits in VI are designated A - G and range in size between 1 1 6 kDa
and 13 kDa (Finbow and Harrison, 1997). Three of these, A, B, and C, have sequence
similarity with a, f!, and y fkom the FiFo-ATPase the others having no obvious relationship.
The stoichiometry of the VI sector is similar to that of the Fi sector, possessing a pseudo-6-
fold symmetry a i s . The Vo sector is composed of three different subunits, two of which are
transmembrane (c and a large glycoprotein) and one which is hydrophilic, but tightly bound
to the integral membrane components (Finbow and Hamson, 1997). Electron microscopy
has shown that the are 6 c subunits in the Vo sector, which are arranged symmetrically
(Holzenburg et al., 1993). The stoichiometry o f the other major subunits is the sarne as in the
FI Fo-ATPase (Nelson, 1992).
The mechanism by which V-type ATPases fùnction is poorly understood at the
moment; however, the recent crystal structure of the FI sector has ailowed a putative
mechanism to be proposed for the F-type-ATPases. Based on their structural similanties,
one would predict the mechanism of action to be quite simiîar for V-type ATPases. The
proposed mechanism is based on a rotating or alternate binding change mechanism. Several
biochemical studies have revealed that ADP + Pi is converted into ATP at two or al1 three of
the highly cooperative binding sites without major input of energy (Boyer, 1993; Boyer et
al., 1973). However, the newly formed ATP molecule remains tightly bound and substrate
binding at another site is coupled to product release at the currently active site. This latter
process requires a major input of energy, which is derived from conformational changes
linked to proton flux through the Fo subunit. A series of elegant experiments have shown
that hydrolysis of ATP at the three catalytic sites in the a3P3 hexamer drives the y subunit
around in three steps (Duncan et al., 1995; Noji et al., 1997; Sabbert et ai., 1997; Zhou el al.,
1996).
In ATP synthesis, this process would operate in reverse, where confonnational
changes in the Fo subunit resulting fiom proton flux would be translated to the y subunit. The
confonnational changes generated by proton binding at the c subunits in the Fo sector are
transmitted to the y subunit in the FI sector via the E subunit. Crosslinking studies have
shown that the E subunit extends the entire length of the s tak region (Aggeler and Capaldi,
1996; Zhang and Fillingame, 1995). The translocation of 4 protons leads to a 120' rotation in
the y subunit, releasing one ATP molecule. III this way, the torque generated in the Fo subunit
by the binding of a protons is transmitted to the y subunit, forcing it to rotate in the center of
the a3 f3; hexamer, releasing new ly synthesized ATP.
1.4.2 Menkes and Wfson Disease Copper Transporiing A TPases
The Menkes (MNK) and Wilson (WND) disease proteins are copper transporting P-
type ATPases which have recently been identified in humans (Bull et al., 1993; Chelly et al.,
1 993; Mercer et ai., 1993; Tanzi et al., 1993; Vulpe et al., 1993). Sequence analysis of these
proteins indicate that they belong to a subfamily of eukaryotic cation transporting P-type
(Type 1) ATPases (see section 1.4.1.1) which is closely related to bacterial heavy metal
ATPases (Bull et al., 1993; Chelly et al., 1993). The Menkes and Wilson proteins share a
high degree of homology with each other. The overall sequence identity between the two
proteins is 57%- however; this figure rises to 79% and higher in the phosphatase,
transduction-phosphorylation, ATP and metal binding domains (Bull et al., 1993). Despite
their similarities, the MNK and WND proteins do have some distinct differences. The
greatest of these is a 78 amino acid deletion in WND between the first and second metal
binding domains (fig. 1.4). The significance of this deletion in the bc t ion of the proteins is
not yet known. interestingly, the rat orthologue of the WND gene is missing metal binding
region 4, but the overall spacing and size of tne amino-terminal metal binding domain is
intact (fige 1.4) (Wu et al., 1994).
The MNK and WND ATPases are type 1, P-type ATPases having several features
which distinguish them fiom other members of the P-type ATPase family. Sorne of these
were discussed in section 1 -4.1.1, including an additional pair of transmembrane segments
immediately before the TGES motif and the absence of four transmembrane segments at the
carboxy-terminus, in cornparison wiîh type II P-type ATPases (see fig. 1.3). In addition to
these differences, the MNK and WND ATPases have a larger amino-terminal domain and
several unique amino acid motifs which are conserved in bacterial heavy metal transporters
(fig 1.5) (Bull et al., 1993; Silver et al., 1993; Tanzi et al., 1993; Vulpe et al., 1993). The
large amino-terminal domain in the MNK and WND protein contains six copies of a metal
binding sequence. The repeat is referred to as the heavy metal-associated (HMA) domain
and has the folIowing consensus sequence: FrVNJ-x(2)-[LIVMFAI-x-C-x-[STAGCDNHI-
C-x(3)-[LIVFG]-x(3)-~JV]-x(9,ll)-~A]-x-[LVFYS]. The highlighted cysteine residues
are strictly conserved in al1 proteins which contain the repeat and they are most likely
involved in the ligation of the metal atom.
The HMA has been found at the amino-terminus of a wide variety of heavy metal
ATPases from various organism involved in the transport of copper, zinc, cadmium and
mercury (table 1.6). The phylogenetic relationship between the proteins listed in table 1.6 is
I Metal Binding
f 1
1 3 ~ 5 6 Td C W h ATP
,- Metai Binding 1 1 2 3 4 5 6 Td C M h ATP
1-' Metal Bindin 1 1 2 3
œ - œ 5 6
m - Td 9
Ch/Ph M
ATP 9
: ,' y: - ç ' .a< J!, y$+'*< y ?x?3 *% < :-:. ;- ,. ;:&!i~ei::~*.:7-k~yf=.-.- \ --,,- +,.. + P. 7- -,-.J$$& . . -... --" -&-,. -Cc?e:Içd.*.n
Figure 1.4 Cornparison of Wilson (WND), Menkes (IWVK) and Rat (rWND) Copper Transporting ATPases. WND contains a large deletion (-78 amino acids) between metal binding repeats 1 and 2. rWND is missing the fourth metal binding repeat but overall spacing is intact. Vertical bars represent transmembrane segments, Td, transduction domain, Ch/Ph, channef phosphorylation domain, ATP, ATP binding domain.
ATX1 ATOXl CopA MerP CadA
ILGMTCQSCVKSIEDRISNLKGIISMKVSL VEGMTCQSCVSSIEGKVRKLQGVVRVKVSL IDGMHCKSCVLNIEENIGQLLGvQS IQVSL IAGMTCASCVHSIEGMISQLEGVQQISVSL IKGMTCASWSNIERNLQKEAGVLSELVAL ITGMTCASCVHNIESKLTRTNGITYASVAL
W-MTCSGCSGAVNKVLTKLEPDVSKIDIS VD-MTCGGCAEAVSRVLNKL- -GGVKYDID ITGMTCANCSARIEKELNEQPGVMSATVNL ITGMTCDSCAVHVKDALEwPGVQsADvCT VQGFTCANCAGKFEKNVKKIPGVQDAKVNF
Figure 1.5 Proposed Structure for Wilson and Menkes Copper A TPuses. A Proposed structure for the Menkes (MNK) and Wilson (WND) disease copper ATPases based on sequence comparison with other cation transporting ATPases. B Aligoment of metal binding domains from WND with other heavy metal transporters. ATXl copper chaperone from S. cerevisiae, ATOX 1 human homologue of ATX 1, CopA copper ATPase from E. hirae, MerP mercury transport protein fiom P. Aeruginosa, CadA cadmium ATPase from S. Atweus. Conserved cysteine residues are bolded. CPC and SEHPL motifs are unique heavy metal transporting ATPases.
Table 1.6 Metai Transporting Proteins Containing Amino-
Terminal HMA Domains
Protein Organism Function EMA Swiss-Prot
YBAR MNK
WND
CCC2
SYNA
CopA
CopP
ATOX 1
ATXI
CUA- 1
ZntA
ZiAA
CadA
Fixi
PacS
CQA CtpB MerA
MerP
E . Coli
Human C. Elegam Rat Mouse
Human Rat Mouse Sheep
S. cerevisiae
S y n e c o c o c ~ ~ ~ 7942
E. Hirae
H . Pylor-i
Human S . cerevisiae
C. Elegans
E . Coli
Synechocystis Sp. PCC 6803
B. Firmus S . A ureus
R. hleliloti
Synechocystis Sp. PCC 6803
M. leprae
M. leprue
P . A emginosa
-
AC # Cu-Transporthg ~TPase ' 1 P78245
Cu-Transporting ATPase Cu-Transporting ATPase Cu-Transporthg ATPase Cu-Transporting ATPase
Cu-Transporting ATPase Cu-Transporting ATPase Cu-Transporting ATPase Cu-Transporting ATPase
Cu-Transporting ATPase
Cu-Transporting A T P ~ S ~ '
Cu-Transporting ATPase
Cu Ion Binding Protein
Copper Chaperone
Copper Chaperone
Cu-Transporting ATPase
Zn-Transporting ATPase
Zn-Transporting ATPase
Cd-Transporting A T P ~ S ~ ' Cd-Transpcrting A T P ~ S ~ '
Nitrogen Fixation
Cation Transp. ATPase
Cation Transp. ATPase
Cation Transp. ATPase Hg@) Reductase
P . Aeruginosa Hg;@) Transport Protein 1 PO413 1 ' ~ h e fourth HMA domains in these proteins are non-hinctional. 'probable function based on primary sequence malysis.
s h o w in figure 1.6. When the HMA dornaia in the CopA protein (E. hirae copper ATPase)
is compared to each of the HMA domains in the WND protein, the degree of similarity rises
from 11% identity for the amino-terminal most HMA domain to 40% identity for the
carboxy-terminal most HMA domain (Petmkhin et al., 1994). The identity between the
carboxy-terminal most HMA domains of MNK and WND is 67%. These observation
suggest that the evolution of the large amino-terminal domain in the MNK and WND
proteins proceeded by gene duplication involving the 'splicing' in of each metal binding
domain to the ATPase core, with the carboxy-terminal most domains being the latest
additions (Petnikhin et al., 1994).
The MNK and WND proteins, as well as al1 bacterial heavy metal transporters,
contain two unique amino acid sequences which are not present in non-heavy metal
transporters, these being the CPC motif in the sixth trammembrane segment and the SEHPL
motif following the phosphorylation site. Particularly interesting is the SEHPL motif which
is absolutely conserved in MNK, WND and bacterial copper ATPase CopA (Odematt el al.,
1993; Petrukhin et al., 1993; Tanzi et al., 1993). This strict evolutionary conservation
suggests that this region is of functional importance, a notion which is strengthened by the
observation that mutation of the histidine residue in this motif to a s e ~ e leads to the Wilson
disease phenotype (Thomas et al., 1995).
Although no specific fùnction has been anributed to this motif in the heavy metal
ATPases, the corresponding region in the sarco(endo)plasmic reticulum Ca-ATPase
(SERCA) is the site of interaction witb phospholamban (James et al., 1989; Toyofiiku et al.,
1993). Phosphotamban is an endogenous, membrane bound inhibitor of SERCA which
interacts with the calcium fkee form of the ATPase and lowers its apparent affhity for
WND (Sheep)
WND (Human)
WND (Rat)
WND (Mouse)
CopA
CadA (B. Fimius)
CadA (S. Aureus)
a
CopP
MerP
Mer A
CCc2
MNK (C. Elaguns)
CUA-1
MNK (Human) m
p SYNA
MNK (Rat)
MNK (Mouse)
L
i
I' YBAR cip.
PacS
Figure 1.6 Phylogenetic Relationship Between HU4 Containing Proteins. Protein sequences obtained from the Swiss-Prot database were aligned using ClustalX with the Blossum 30 amino acid weighting matrix. The phylogenetic tree was calculated using ClustalX. See table 1.6 for Swiss-Prot accession numbers for each protein.
calcium. As calcium concentrations rise and more of the enzyme is converted to the calcium
bound form, phospholamban is released fkom the ATPase. Phospholamban could also be
released from the ATPase through phosphory lation of p hospholamban by c AMP-dependent
protein kinase A (Toyofuku et al., 1993). In the plasma membrane calcium ATPases, the
analagous region was shown to interact with a synthetic calmodulin binding domain
(FaIchetto et al., 1991). The interaction of calmodulin with the plasma membrane calcium
ATPase has been shown to resuit in an approximate 20-fold increase in the affinity for
calcium (Larsen et al., 1981; Larsen and Vincenzi, 1979). In Na-/K- ATPases this region is
susceptible to proteolytic digestion in the presence of potassium, but resistant to digestion
when sodium is present (Jorgensen and Farley, 1988). These fmdings suggest that, in these
ATPases, this region undergoes conformational changes during the transport cycle of the
protein. The amino-terminal metal binding domains could play a regulatory role in addition
to binding copper. In the absence of copper, the metal binding domain may interact with the
SEHPL containing segment; once copper binds, conformational changes would release this
interaction, activating phosphorylation of the enzyme and starting the transport cycle.
As compared to non heavy-metal transporting P-type ATPases, the WND and MNK
proteins have two less transmembranç segments at the carboxy-terminus. In both the
SERCA and Na-/KT ATPases the carboxy-terminal transmembrane segments are intimately
involved in the cation translocation process (Clarke et al., 1989; Karlish et al., 1990). In
addition it is interesting to note that the KdpK7-ATPase in E. coli also lacks these carboxy-
terminal transmembrane segments and requires other subunits to form a fùlly functional
complex (see section 1.4.1.1). These observations suggest the possibility that the WND and
MNK proteins require a second, unidentified subunit. This type of arrangement is seen in the
cadmium efflux systern of S. aureus in which the components of the cadmium resistance
mechanism (cadA and cadB) reside on two separate plasrnids (Nucifora et al., 1989). The
CPC motif present in the sixth trammembrane segment is also strictly conserved between the
MNK, WND and bacterial heavy metal transporting AïTases. The proline residue in this
motif is conserved in ail P-type ATPases and has been shown to be critical in the formation
of high affinity calcium binding sites in the SERCA ATPase (Vilsen et al., 1989). It is likely
that the conserved cysteines in this motif are involved in binding the copper atom transiently
as it moves through the membrane.
The WND and MNK proteins also have some important structural differences when
compared to closely related bacterial heavy metal transporting ATPases. First, the MNK and
WND proteins contain six copies of the HMA domain at their amino-terminus, while only
one or two copies are present in the analogous prokaryotic proteins (Bull et al., 1993. Tanzi
et al., 1993). Second, the WND and MNK proteins contain a long carboxy-terminal
hydrophilic fragment which is similar to that found in the plasma membrane calcium ATPase
and the H- ATPase in plants (Pardo and Serrano, 1989). Lastly, the length of the sequence
between the phosphorylation site to the hinge domain is 70 - 80 amino acids longer than in
the analogous bacterial ATPases, but substantially shorter than in the corresponding region of
other eukaryotic ATPases, As discussed above, this region in eukaryotic caIcium ATPases is
the site of interaction with phospholamban and calmodulin, two proteins which regulate the
function of these ATPases. In contrast, the malarial calcium ATPase is lacking this
sequence and, in its place, has an additional 100 - 200 amino acids which are not found in
vertebrates, suggesting that this ATPase has its own regulatory protein (Kimura er al., 1993).
By anaiogy, it has been suggested that the additional 70 - 80 amino acid residues carboxy-
terminal to the SEHPL motif in WND and MNK may reflect the unique regdatory
requirements of these proteins (Petrukhin et al., 1994).
1.5 Human Genetic Disorders of Copper MetaboUsm
Although trace arnounts of many transition metal elements are needed to sustain life,
arnounts in excess of what is needed are usually detrimental. Therefore, precise control over
the free concentrations of these trace metals is essential to maintain good health. This is
especially important for redox active rnetals such as iron and copper which, if present in
excess, can enhance free radical geaeration and lead to cellular damage (Britten, 1996). In
humans, the two major genetic disorders of copper metabolism are Wilson and Menkes
disease which are characterized by an excess and a deficiency of copper respectively. Recent
advances in the cloning of the genes responsible for these disorders has led to several major
insights into copper transport and intracellular trafficking.
1.5.1 Menkes disease
1 .S. 1.1 Genetics of Menkes Disease
Menkes disease (OMIM 309400) is a genetic disorder of copper metabolism
characterized by an impairment in the absorption of dietary copper and a severe disturbance
in the intracellular transport and trafficking of copper (Danks et al., 1973; Herd et al., 1987).
Menkes and coworkers noted in their original study that the disorder seemed to be confined
to males and was inherited in a sex-linked recessive marner (Menkes et al., 1962). The
incidence of Menkes disease is approximately 1/300,000 live births (Tümer and Hom,
1997b). Early work by a number of researchers mapped the disease locus to band Xq13.3
(Tümer er al., l992b; Tümer et al., 1992a; Verga et al., 1991). Using this early genetic work
as a guide, the Menkes disease gene (ATP7A) was isolated independently by three groups
using positional cloning techniques (Chelly et al., 1993; Mercer et al., 1993; Vulpe et al.,
1993). The gene was found to contain 23 exons which span approximately 150 kb of
genomic DNA (fig. 1.7) (Dierick et al., 1995; Tümer et al., 1995). The exons range in size
from 77 to 726 bp, with introns ranging fiom 60 to 196 kb (Dierick et al,, 1995). Al1 splice
sites were found to conform to the GT-AG rule, except for the donor site of intron 9, which
precedes an exon which is altematively spliced (Dierick et al., t 995). The Menkes gene
(ATP7A) shares some similarity with Wilson disease gene (ATP7B). Following the exon
coding for the fifth metal binding domain (exon 5 in ATP7A and exon 3 in ATP7B), the
coding region of both the Menkes and Wilson proteins are virtually identical (Tiimer er al.,
1995). However, significant differences do occur at the 5' end; where the first four metal
binding domains in the Wilson disease gene are contained in one exon, those of the Meakes
disease gene extend over three exons (Dierick et al., 1995).
The Menkes gene encodes a 1500 amino acid protein which is predicted to be a
copper transporting P-type ATPase. The predicted protein has a high degree of similarity to
that encoded by the Wilson disease gene (see section 1.4.2). The main difference is the
presence of a 78 amino acid insertion between metal binding domains 1 and 2 in the Menkes
protein which is not present in the Wilson disease protein. Northem blot analysis has shown
that the Menkes disease protein is expressed in al1 tissues except the liver. which correlates
welI with the observed clinical and biochemical features of the classical Menkes phenotype
(Cheiiy et al., 1993; Vulpe et al., 1993). Analysis of numerous Menkes cases has revealed
that approximately 15-20% of the mutations found in the Menkes disease gene consist of
- Transmembrane segments
CU - Copper binding repeats
PD - Phosphatase dornain
C - Conserved CPC motif
D - Pbosphorylation domain
A - ATP binding domain
Figure 1.7 Genomic Structure of the Diseuse Gene (ATP7A). Vertical lines indicate the positions o f the introns, the numben indicate the positions of the exons. Adapted fiom (Tümer and Hom, 1997b).
CU CU CU CU CU CU #Ru #
I I I I I I l I I I / I l I I I I I I I I I I I
7 8 9 1 2 1 0 1 11192021 22 23 5 6 3 4
insertions or deletions of varying sizes (Chelly et al., 1993; Mercer et al., 1993; Vulpe et al.,
1993). In a study of 41 unrelated patients presentiag with the ciassical form of Menkes
disease, 1 9 out of 4 1 mutations were due to insertion or deletions (Tiimer et al., 1997a). The
two Iargest of these deletions are a 14 bp deletion in exon 10 and a 10 bp deletion in exon 22.
These deletions and others are predicted to alter the reading h m e of the gene, leading to the
introduction of a stop codon and premature truncations of the protein. Alternatively, these
mutations may lead to the skipping of subsequent mutation-bearing exons, resulting in an
internally deleted protein which may retain some function as has been reported for a 2 bp
deletion in exon 7 @as er al., 1994).
The snidy by Tümer el al. (Tümer et al., 1997a) also discovered that approximately
half of the mutations they identified were Iocated in exons 7-10 and half of these mutations
affected exon 8 alone. Furthemore, five of the twelve point mutations identified in another
series also affect exon 8 (Das et al., 1994). Exon 8 encodes a region of the protein between
metal binding domain 6 and the first transmembrane segment of the protein. Although no
specific function has been attributed to this region in the protein, it may play a role in the
precise localization of the metal binding domain relative to the ATPase core. The
importance of this region of the protein is demonstrated by the finding that individuals
carrying mutations in exon 8 prescnt the classical severe form of Menkes disease (Tümer et
al., 1997a). In contrast, only two mutations have been found in the corresponding region of
the Wilson disease gene (Figus et al., 1995). The vast difference in the fiequency of exon 8
mutations between the two genes could indicate that, despite their sirnilarities, these proteins
may have significant differences in their mechanism of action.
1 .S. 1.2 Clinical and Biochemical findings
Menkes disease was originally described by John Menkes and coworkers in 1962
during their study of five patients from the same family who al1 died before uiree years of
age (Menkes et al., 1962). Patients affected by the classical severe form of the disease
originally described by Menkes and coworkers are unable to absorb copper and hence suffer
from the effects of copper deficiency, As observed by Menkes, Menkes disease has a very
early age of onset with untreated patients usually dying before the age of three. The patients
in the original study were al1 delivered nonnally and had normal birthweights. However, in
the weeks following birth, several phenotypes were observed wbich have now become the
hallmarks of Menkes disease. It was noted that al1 patients gained very little weight despite a
normal diet, and al1 had hair abnormalities either from birth or developing soon thereafter.
These abnormalities consisted of hair that was coarse and brinle and was white in color due
to depigmentation. Microscopic examination of individual hair fibers revealed several gross
abnormalities, they were either twisted (pili torti), Eractured at various intervals (trichorrexis
nodosa), or of varying caliber (monilethrix). However, despite these abnormalities, the level
of sulfhydrys in the hair were found to be normal (Menkes et al., 1962). The presence of
these haïr abnormalities in Menkes patients Ied to the disorder also being referred to as
kinky-haïr syndrome.
Other case studies in the years following Menkes' initial description of the disorder
expanded the list of clinical symptoms (Table 1.7) and have identified a moderate fonn, a
late onset form, and a mild allelic form (occipital hom syndrome, OHS) of the disorder
Table 1.7 Symptoms of Classical Menkes Disease and
Occipital Horn Syndrome (OHS)
Classical Menkes OHS Connective Tissue Symptoms
Tortuous vessels + + Skeletal changes + ++ B ladder diverticulae t ++ Loose skin + -I- Loose joints + +
Neurologie Symptoms Mental retardation Convulsions Hypo thennia Feeding dificulries Muscle tone changes
O ther Symptoms Facial dysmorphism Abnonnal hair Hypopigmentation
Laboratory Findings Serum copper 4 4 Semm ceruloplasmin 4 4 Intracellular copper accumulation +
Life Expectancy <3 years NK' Adapted from (Tümer and Hom, 1997b). ' ~ i f e expectancy for OHS patients is not known. AI1 known OHS patients are still alive with the oldest being 53. N D = Not Detennined, NK = Not Known.
(Danks, 1988; Danks et al., 1972; Hom et al., 1995; Peltonen et al., 1983; Procopis et al.,
1981). Although most patients with OHS have only mild deficiencies, several cases with
more severe symptorns such as psychomotor retardation and seizures have been described
(Mentzel et al., 1999; Wakai et al., 1993). The patients were also found to have extremely
low levels of copper in the senun, brain, and liver, and extremely low ceruloplasmin levels.
Typicai copper measurements fiom Menkes patients are summarized in table 1.8. Attempts
to correct the problem by oral administration of copper failed; however, intravenous injection
of copper was able to nonnatize ceruloplasmin levels and copper clearance fiom the serurn
was also norrnalized (Danks et al*, 1972). This and other evidenced strengthened the idea
that the basic biochemical defect was a severely compromised ability to absorb intestinal
copper, compounded by disturbances in the intracellufar transport and traficking of copper
(Danks et al.. 1973; Herd et al., 1987). Cultured fibroblasts fkom Menkes patients have been
found to accumulate copper in the f o m of copper metallothionein and metallothionein
induction in cultured muscle cells from these patients is elevated relative to normal ceIls
(Beratis et al., 1978; Chan et al., 1978; Goka et al., 1976; Herzberg et al., 1990; Onishi et al.,
1 980; Riordan and Jolicoeur-Paquet, 1 982).
The clinical symptoms of classical Menkes disease can be attributed to a deficiency
of developmentally important cuproenzymes such as lysyl oxidase, tyrosinase, cytochrome c
oxidase, dopamine fi hydroxylase, CdZn superoxide dismutase and amine oxidase (table 1.3)
(Danks, 1995; Menkes, 1988). Lysyl oxidase is an essential enzyme needed for the
crosslinking of connective tissue; a deficiency in this enzyme would lead to weakened
comective tissue, which could lead to the comective tissue defects, such as arterial rupture,
Table 1.8 Typical Copper Measurernents in Normal Adults and
Menkes Disease Patients
Menkes Patient Normal Infant (3-12 Months) (3-12 Months)
Serurn ceruloplasmin OD unitdm1 ~0.08 >0.25 mgk <50 >200
Semm copper (pM) <6 >12
Liver copper (pg/g dry weight) 10 - 20 70 - 140
Duodenal copper (pg/g dry weight) 50 - 80 7 -29 Adapted fkom (Danks, 1995).
observed in Menkes patients (Danks, 1995; Kuivaniemi et al., 1982; Peltonen et al., 1983).
The absence of tyrosinase could lead to the hair depigmentation obsewed in Menkes patients
and low levels of cytochrome c oxidase can result in temperature instability and seizures
(Bankier, 1995). Examination of the cuproenzyme status in hepatocytes fiom murine models
of Menkes disease reveals that they have normal activity, although the level of copper in the
liver is severely depressed (Phillips et al., 1986). This suggests that intracellular copper
transport in the liver is normal in Menkes patients.
The biochemical effects of Menkes disease suggest that the body is in a condition of
copper deficiency. Therefore, the Menkes ATPase would be expected to be present in the
intestinat mucosa where it would function to transfer copper fiom the intestine into the blood
Stream. In Menkes disease, the ATPase is non-functional, resulting in an accumulation of
copper in the intestines in the f o m of copper metallothionein, which is lost fiom the body
when these cells are sloughed off leading to a global copper deficiency. Recent studies using
immunofluorescence techniques have localized the Menkes ATPase to the golgi apparatus
under normal copper concentrations (Petris et al., 1996; Yamaguchi et al., 1996). It has also
been shown that under elevated copper conditions, the ATPase undergoes a reversible,
copper dependent relocalization corn the golgi to the plasma membrane (Petris et al., 1996).
Therefore, under normal copper conditions, the ATPase functions to pump copper into the
golgi for incorporation into cuproenzymes or to pump copper into the body from the
intestinal rnucosa. When elevated copper conditions are encountered, the ATPase is
translocated to the plasma membrane where it functions to expel copper fiom the cell. These
observations explain why fibroblasts fiom Menkes patients have nomai copper uptake
activity, but are not able to expel copper to avoid copper accumulation.
1 S. 1 -3 Current Treatmunts
Since the main problem in Menkes patients is a global deficiency of copper,
treatments have concentrated on restoring normal copper levels in the body by the
administration of copper. However, since intestinal absorption of copper is extremely low in
Menkes patients, the copper complexes must be administered parenterally. Some early
complexes which were evaluated for treatment purposes included copper chloride, copper
sulfate, copper EDTA, and copper albumin. However, it was found that these complexes are
unable to produce significant clinical improvement especially in regards to
neurodegeneration (Garnica, 1984). The only copper complex which has been used
successfully to treat Menkes disease is copper-histidine (Sarkar, 1980). Copper-histidine is a
nonnal component of human serum and has been shown to play an important rote in the
transport and uptake of copper (Mas and Sarkar, 1992; Sarkar, 198 1 ; Sarkar and Kruck,
1966). Detailed copper uptake studies conducted in vitro have suggested that copper uptake
by brain tissue is mediated by complexation to histidine and have also suggested that the
copper-histidine complex is abte to cross the blood-brain barrier (Bamea et al., 1990; Barnea
and Katz, 1990; Hartter and Barnea, 1988; Katz and Bamea, 1990).
Patients treated with subcutaneous injections of copper-histidine have shown
significant clinical and biochemical improvement (L-euder et al., 1993; Sarkar et al., 1993;
Sherwood et al., 1989). The best outcomes have occurred in patients in which treatment was
started as early as possible. These case studies have s h o w a normalization of serum copper,
ceruloplasmin, dopamine and norepinephrine lrvels after three months of treatment (Kreuder
et al., 1993). The norrnalization of dopamine and norepinephrine levels indicate that nonnal
dopamine fLhydroxylase levels may have been restored. The severity of the mutation in the
two longest lived Menkes patients who have responded very well to copper-histidine
treatrnent bas been confirmeci by DNA sequence analysis. It has been discovered that these
two patients harbor mutations in the Menkes disease gene which totally abolish the
production of a functional ATPase. In the first patient a mutation in exon 4 introduces a
premature stop codon in the third metal-domain, resulting in the production of a polypeptide
without the ATPase 'core'. In the second patient the mutation occurs in exon 12, again
leading to the introduction of a stop codon upstream of the phosphatase domain. This
mutation produces a polypeptide which contains the first four traasmembrane segments, but
Iacks the remainder of the ATPase 'core' (Tiimer et al., 1996).
Despite significant clinical and biochemical improvements in these patients,
comective tissue disorders continue to persist, indicating that lysyl oxidase levels are not
being restored by copper-histidine injections. Altematively, the persistence of these
disorders rnay reflect the slow turnover of comective tissues. It is also likely that copper
presented in the copper-histidine complex is not available for incorporation into lysyl
oxidase. Studies camed out in brindled mice, a murine mode1 for Menkes disease, have
shown that subcutaneous injections of copper(i) in an alkyl polyether / sebacic acid solution
are able to increase dramatically the level of lysyl oxidase in these animals (Royce et al.,
1982). These findings suggest that the oxidation state of copper may influence its ability to
penetrate certain intracellular compartments. Hence the treatment of Menkes patients could
be enhanced by providing copper both in the +1 and +2 oxidation state, possibly using
copper-histidine in conjunction with another agent.
1 .S. 1.4 Animal Modeis of Menkes Disease
Animal models are indispensable tools for the study of hurnan disorders since they
permit studies which would otherwise be impossible in humans. For Menkes disease the
main animal models are the mottled mutations in mice (Danks, 1977; Danks, 1986; Danks
and Camakaris, 1983; Hunt, 1974; Xu et al., 1994). A cornparison of the symptoms found in
these animals with Menkes patients is shown in table 1.9 The series of mutants, mottled
(Mo), dappled (MO~P), tortoiseshell (Mo'"), brindled (Mobr), viable brindled ( ~ 0 ~ ~ 3 , blotchy
MO^"), macular ( ~ 0 ~ ' ) and pewter (MoPe') are usually considered allelic and display
extensive phenotypic variability. These series of mutations were identified in the 1950s as
X-tinked mutations which led to patchy coat color in females and were lethal in males. In
hemizygous males the displayed phenotypes include connective tissue disorders in blotchy
and viable brindled, prenatal lethality in dappled and tortoiseshell, severe neurologie disease
in brindled and macular, and isolated coat color defects in pewter (Levinson et al., 1994).
Early studies on the brains h m brindled males revealed that copper levels were
significantly decreased (Hunt, 1974). This led to the proposa1 that a deficiency of copper
was the primary defect in these animals and the diverse phenotypes arose fkom secondary
deficiencies in copper-requiring enzymes (Darwish et al., 1983). This idea was further
strengthened by the finding that cultured cells k o m many organs, except the liver, had
defects in copper export, which resulted in the cytosolic accumulation of copper (Dawish et
al., 1983; Packman, 1987; Sayed et al., 198 1). Defective export and accumulation of copper
are also found in cultured fibroblasts from Menkes patients (Beratis et al., 1978; Chan et al.,
1978; Goka et al., 1976; Onishi et al., 1980). The mouse orthologue of the human Menkes
Table 1.9 Features of Menkes Disease in Humans and the
Mottled Mouse Mutants
Menkes Brindled Blotchy Disease Mouse Mouse
Hair deforrnity +t + +
Hair keratin disulfide deficiency + + 4-
Arterial abnonnalities f+ + / - -+
Lysyl oxidase deficiency + + +
Li fe expectancy 1-2 years 13- 15 days 6- 18 months
Liver copper J. J. J.
Kidney copper 'r + It‘
Intestinal copper 'r 'r 'r Adapted fiom (Danks, 1995).
gene has been cloned, and resides on a region of the mouse X-chromosome which is syntenic
to the region where the human Menkes gene is located (Cecchi and Avner, 1996; Levinson et
al., 1994; Mercer et al., 1994).
1 S.2 Wilson disease
1 S.2.l Genetics of Wilson Disease
Wilson disease (OMIM 277900) is an autosomal recessive disorder of copper
transport which was first described by Kimear Wilson in 19 12 (Wilson, 1912). Wilson
disease is characterized by the toxic accumulation of copper in various tissues but primarily
the liver, kidney, and brain. The fiequency of the disease is approximately 1/100,000 but
seems to be more prevalent in Japan, Sardinia, and China (Reilly er al., 1993; Sternlieb,
1990; Türner and Horn, 199%). Early genetic studies assigned the Wilson disease gene to a
single locus by linkage to the esterase D gene (Bull et al., 1993; Frydman et al., 1985) while
Iater studies placed the gene close to a cluster of polymorphic markers in the q14.3 band of
chromosome 13 (Bowcock et al., 1987; Bowcock et al., 1988; Yuzbasiyan-Gurkan er al.,
1 988). Following these studies, the Wilson disease gene (ATP7B) was cloned independently
by two groups (Bull et al., 1993; Tanzi et al., 1993). The Wilson disease gene consists of 22
exons (fig. 1 .a), with exon 2 1 being expressed in the liver and exon 22 being expressed in the
kidney (Cox, 1995; Petrukhin et al., 1994; Thomas et al., 1995). The exons range in size
from 77 bp to 2355 bp (Petrukhin et al., 1994). The ATP7B transcript is expressed at a high
level in the liver and kidney, with a lower level being detected in the lung and placenta (Bull
el al., 1993). The pattern of expression correlates well with the observed clinical and
biochemical features of the disease (see section 1.5.2.2). The gene is predicted to encode a
- Transmernbrane segments
Cu - Copper binding repeats
PD - Phosphatase domain
C - Conserved CPC motif
O - Phosphorylation domain
A - ATP binding domain
1
Figure 1.8 Genornic Structure of the Wilson Disease Gene (ATP7B). Vertical lines indicate the positions of the introns, the numbers indicate the positions of the exons.
I I I I I I I I I I I I I I I I I I I I I
2
Cu Cu Cu Cu Cu Cu 1 I 3 5 6 7 4 8 910111213141
copper transporting P-type ATPase which has many features in common with other cation
transporting P-type ATPases as well as several bactenal heavy metal transporters (see section
1.4.2).
In contrast to the Menkes gene, most of the mutations in the Wilson disease gene do
not involve large insertions or deletions. Missense mutations in the Wilson disease gene
have been found in al1 exons, with the exception of exons 1-5, which encode the six amino-
tcrminal metal binding motifs, and exon 21, which encodes the carboxy-terminal tail of the
protein (Shah et al., 1997; Thomas et ai., 1995). Many other mutations in the Wilson disease
gene, including small insertioddeletions, nonsense, hmeshifi, and splice site mutations have
been found and are summarized elsewhere (Chuang et ai., 1996; Loudianos et ai., 1996;
Nanji et al., 1997; Shimizu et al., 1995; Thomas et al., 1995). From these studies, it is clear
that the most common missense mutation in many populations is His1069Gln which is
present in about 38% of homozygous Wilson disease patients. The mutation occurs in a
conserved Ioop motif (SEHPL), which is adjacent to the conserved phosphorylation motif
DKTG. No specific function has yet been attributed to this motif, but it seems critical for the
correct fiinction of the heavy metal ATPases, as it is absolutely conserved in the Wilson,
Menkes, and CopA (bacterial copper ATPase) proteins (Odermatt et al., 1993; Petrukhin et
al., 1993; Tanzi et al., 1993).
The high fiequency of the His 1069Gin mutation has permitted a genotypelphenotype
correlation for this mutation. In one study, patients hornozygous for the His1069Gln
mutation were found to have a later average age of onset as compared to the heterozygous
patients, 20 years versus 15 years (Shah et al., 1997). In addition, al1 homozygotes were
found to have an equal incidence of bepatic and neurologic symptoms. The His1069Gln
mutation was also found at high fiequency in a study conducted on Mediterranean
populations (Figus et al., 1995). This study also found that the Hisl069Gin is always
associated with the same haplotype suggesting that it most likely arose from an ancient
mutational event (Figus et al., 1995). The molecular basis of the adverse effects of the
His1069Gln mutation has recently been investigated. Recent studies have s h o w that
transfection of the Wilson cDNA containhg the His 1069Gh mutation into mouse fibroblasts
gives rise to a protein with a 5-fold shorter half-life, which is mislocalized to the endoplasmic
reticulum (Payne et al., 1998a). The authors also noted that the mislocalization was
temperature-sensitive and suggest that misfolding followed by degradation constitutes the
molecular basis of the defect in these Wilson patients.
1 .S. 2.2 Ciinical and Biochemicaljïndings
Wilson disease results fiom the accumulation of toxic levels of copper in various
organs due to a severe deficiency in the excretion of copper fiom the body. Wilson disease
displays extensive clinical heterogeneity and has a variable age of onset which can range
from the age of 8-9 years to the mid-50's. The progression of the disease can be roughly
divided into three distinct clinical phases. in the first phase, copper begins to accumulate in
the cytoplasm of hepatocytes due to impaired biliary excretion of copper (Strickland et al.,
1973). In the second phase, as more copper is absorbed, the cytosolic concentration of
copper rises resulting in the induction of large amounts of metallothionein and to the
chelation and storage of excess copper (Hunziker and Stemlieb, 1991). The high levels of
cytosolic copper in hepatocytes eventually leads to necrosis, resulting in the release of large
amounts of copper into the bloodstream. In the final stage of the disease, increasing arnounts
of copper begin to accumulate in other organs sucb as the brain, kidney and comea. The
sudden influx of large amounts of copper into the bloodstream results in oxidative damage to
erythocyte membranes, leading to hemolytic anemia @obyns er al., 1979; Mowat, 1994).
Wilson disease patients can also be divided into one of three groups, depending on what
combination of symptoms are present. Patients may either display hepatic symptoms,
neurologic symptoms, or both.
The deposition of copper in the comea gives rise to duil-yellow Kayser-Fleischer (K-
F) rings which are a diagnostic hallrnark of Wilson disease (Scheinberg and Stemlieb, 1984).
The presence of K-F rings has been found in 95% of a11 Wilson disease patients and in al1
patients which display neurologic disorders (Danks, 1995). As a result of its prevalence in
the majority of patients, K-F rings have become a diagnostic hallrnark of the disease in the
latter stages. The deposition of copper in the brain during the latter stages of the disease
correlates with the finding that patients with an early age of onset usually display
predominantly hepatic symptoms, while those with a later stage of onset display
predominantly neurologic symptoms (S trickland et al., 1 973 ; Vulpe and Packman, 1995).
The list of symptoms relating to hepatic dysfunction is extensive and varied. These include
jaundice, asyrnptomatic cirrhosis, subacute hepatitis (non-A or B) and chronic aggressive
hepatitis (Mowat, 1994). The neurologic syrnptoms in Wilson disease patients c m include
deteriorating coordination, tremors, dysartbria, drooling, personality changes, dementia,
slurring of speech, ngidity and behavioral problems (Mowat, 1994; Strickland and Leu,
1975). The neurologic symptoms in Wilson disease can be attributed to cerebellar or
extrapyramidal involvement or a combination of both (Dobyns er al., 1979).
The biochemical findings in Wilson disease patients are consistent with a disturbance
in the excretion of copper and, pnmarily, a defect in the biliary excretion of copper
(Strickland et al., 1973). Typical copper measurements in Wilson disease patients are
summarized in table 1.10, along with those in normal adults. Urinary copper levels in
Wilson disease patients are elevated due to the accumulation of copper in the kidneys, which
leads to renal dysfunction in the form of albuminuria and renal rickets (Walshe. 1989). Since
incorporation of copper into cerulopIasmin is also impaired in Wilson disease, there is a
greater proportion of copper bound to albumin and low molecular weight complexes in the
serurn; however, the overall serum concentration of copper is lowered (Barrow and Tanner,
1988). Recent immunohistochemical studies have showm that the Wilson disease ATPase is
Iocalized to the tram-golgi network under steady-state conditions (Hung et al., 1997; Nagano
et ai., 1998). Furthemore, as also observed for the Menkes disease ATPase, these studies
have shown that the ATPase undergoes a rapid, copper dependent relocalization fiom the
golgi to a cytosolic vesicular cornpartment, as well as to the plasma membrane.
The function of the ATPase in the trans-golgi is most likely to transport copper fiom
the cytoplasm into this cornpartment for incorporation in secreted, copper-containing proteins
such as ceruloplasmin. This idea correlates with the finding of low ceruloplasmin levels in
Wilson disease patients. The relocalization of the ATPase to a vesicuku compartment or the
plasma membrane in response to elevated copper levels would lead to efflux of copper fiom
the ceI1, either via the bile (through the fusion of vesicles with the canalicular membrane) or
across the plasma membrane (Dijkstra et al., 1996; Hung et al., 1997). There is also one
recent report which has localized the Wilson disease ATPase to the mitochondria, where it
Table 1.10 TypicaI Copper Measurements in Normal Aduits and
Wilson Disease Patients
Wilson Disease Normal Adult Serum ceruloplasmin OD unitdm1 O - 0.25 0.25 - 0.49 mg/L O - 200 200 - 400
Serum copper (CIM) 3 - 10 1 1 -24
Urinary copper (&24 hours) 100 - 1000 40
Liver copper (pg/g dry weight) 200 - 3000 20 - 50 Adapted fiom (Danks, 1995).
has been suggested to provide copper for incorporation into cytochrome oxidase; however,
this bas yet to be coa fmed by others (Lutsenko and Cooper, 1998).
1.5.2.3 Current Treatments
Treatments for Wilson disease patients have concentrated on the use of chelators to
mobiIize copper fkom the centers of accumulation and promote its excretion from the body.
The first chelator used widely for the treatment of Wilson disease was BAL (2,3-
dimercaptopropanol) penny-Brown and Porter, 195 1). in this initial study of five patients,
the authors observed a significant reversa1 of tremors and rigidity following treatment with
BAL. However, these improvements were only observed afier longterm use and were less
significant in patients with more acute cases of the disease (Cartwright et al., 1954; Matthews
et al., 1952). Furthermore, patients had to deal with painful intramuscular injections and
often side effects. These concems led to the development of D-penicillamine which
improved upon BAL in every respect (Walshe, 1956). It could be administered orally, is less
toxic and more effective than BAL. The standard dose of D-penicillamine for adults is 500
mg given twice daily, with close monitoring of urinary copper. On this dose, urinary copper
excretion can increase to between 4 and 5 mg per day (Sass-Kortsak, 1975).
Treatment with D-peaicillamine usually lasts for a lifetime and produces significant
clinical and biological improvements in a large number of cases. In some patients,
neurologie syrnptoms may disappear, liver fùnction may retum to normal, and Kayser-
Fleischer rings usually fade and then disappear (Sass-Kortsak, 1974). However, despite its
vast irnprovement over BAL, some severe side effects have been reported for D-
penicillarnine as weli. The most common side effects in the early stages of treatrnent include
fever, anorexia, pyridoxine deficiency, and a mild depression of leukocyte and platelet counts
(Mowat, 1994; Sass-Kortsak, 1975; Scheinberg, 1968). In very few cases, serious toxic
effects may include systemic lupus erythematosus, and Goodpasture's syndrome (Mowat,
1994; Sass-Kortsak, 1975). D-penicillamine has also been linked to changes in the
crosslinking of collagen and elastin, leading to serious skin and c o ~ e c t i v e tissue disorders
(Iozurni et al., 1997; Narron et al., 1992; Pasquaii-Ronchetti et al., 1989). There are also at
least w o cases on record of asymptomatic Wilson disease patients experiencing severe
newological deterioration soon after initiation of D-penicillamine therapy (Brewer et al.,
1994; Glass et ai., 1990). There is also a danger in intemipting D-penicillamine treatment
once started. There are several cases on record in which patients have stopped taking their
tablets and irreversible liver damage has developed within two years (Scheinberg et al.,
1987; Walshe and Dixon, 1986).
A number of other chelating agents have been developed and used successfully in
cases where patients develop toxic reactions to D-penicillamine or treatment is intermpted.
These inctude trientine (triethylenetetramine dihydrochloride) (Scheinberg et al., 1987;
Walshe, 1 982), tetrathiomolybdate (Brewer et al., 1994a; Walshe, 1984), and zinc therapy
( Alexiou et al., 1 985; Hoogenraad, 1998; Hoogenraad et al., 1979; Hoogenraad et al., 1978;
Van Caillie-Bertrand et al., t 985). Trientine may be a viable alternative to D-penicillamine
in some cases; no toxic side effects have been observed in 20 patients using the drug for up to
13 years (Scheinberg et al., 1987; Walshe, 1982). Trientine has also been employed
successfully in one female patient who had previously been on D-penicillarnine. Trientine
was administered during pregnancy without adverse affects on the fetus (Devesa et al.,
1995). Trientine may be an effective treatment when D-penicillamine has been withdrawn;
however, commercial availability of the compound is still a problem. in addition, trientine
and D-penicillamine seem to act on different pools of copper. D-penicillamine is able to
mobilize copper from the liver, but cornpetes relatively poorly for copper bound to albumin
(Sarkar et al., 1977). In contrast, trientine cannot mobilize copper €rom the liver, but is
effective in competing for copper bound to albumin (Satkar et al., 1977). Both dnigs are
able to mobilize copper fiom the kidneys, promoting its removal through urinaq excretion.
Tetrathiomolybdate is another chelator with great potential which has been used to
treat Wilson disease. It is an extremely potent chelator of copper which can cause severe
copper deficiency in normal rats within 1 week and is, therefore, not suited for long-tenn
treatment of Wilson disease (Brewer et al., 1991; Mason et al., 1989; Walshe, 1989).
However, tetrathiomolybdate can be used for initial decoppering followed by maintenance
with zinc. At the very least, tetrathiomolybdate is a possible alternative in cases where
toxicity to D-penicillamine develops. Unlike other chelating agents which promote the
mobilization and excretion of copper through the urine, zinc therapy promotes fecal copper
excretion. Oral zinc therapy is thought to promote copper excretion by inducing the
synthesis of metallothionein in the intestine, thereby blocking the absorption of copper
(Hoogenraad, 1998; Hoogenraad et al., 1979; Hoogenraad et al., 1978). Although zinc has a
greater ability to induce metallothionein synthesis than does copper, metallothionein has a
higher affinity for copper than zinc (Lipsky and Gollan, 1987). As a result the newly
synthesized metallothionein sequesters copper in the upper intestine, preventing its transport
into the portal circulation. The sloughing off of intestinal cells into the lumen results in the
elevated copper levels observed in the feces of zinc-treated Wilson patients (Brewer et al.,
1983; Lipsky and Gollan, 1987). Patients with acute liver failure and encephalopathy are
usually beyond the help of chelation therapy. Ln these cases liver transplantation may
provide the only hope for survival. The results of 57 Wilson disease patients who underwent
liver transplantation showed chat there was a 72% survival rate after the first year, with 4
patients requiring retransplantation (Mowat, 1994; Schilslq et al., 1994).
1 -5.2.4 Animal Models
Several animal models for Wilson disease have been identified and al1 display clinical
and biochemical symptoms which are very close to those found in human Wilson disease
patients. These animal models include the toxic miik (tx) rnouse (Rauch et al., 1986; Rauch
and Wells, 1999, the Long Evans Cimamon (LEC) rat (Li et al., 1991) and bedlington
temers (Su et al., 1982; Twedt et al., 1979). The biochemical features of these animals, as
compared to Wilson disease, are summarized in table 1.1 1. The toxic milk mouse and the
LEC rat display very similar clinical and biochemical features. The toxic milk mutation was
first recognized as an autosomal recessive trait which was lethal to the pups of affected
females due to a severe copper deficiency in their milk (Rauch, 1983). In affected fernales,
copper accumulates in the liver, leading to histological changes and cirrhosis in the absence
of any neurological effects. It has been shown that the excess copper which accumulates in
the toxic mice is in the fonn of copper metallothionein (Koropatnick and Cherian, 1993;
Rauch et al., 1986).
Table 1.1 1 Cornparison of Biochemical Findings in Wilson Disease and its
Animal Models: Toxic Milk Mouse and Bedlington Temers
Wilson Bedlington Toxic Milk Disease Terrier Mouse
Hepatic copper accumulation +t -ii+ ++ dg dry weight) 200-3000 2000-12000 300-400
Reduced biliary copper excretion f.t + NK
Reduced copper incorporation into ceruloplasmin fft f+ $-t.
Liability to hemolytic crisis + ++ +
Chronic Iiver ceIl damage -+-t + / - tt-
Copper overflow to other tissues ++ + / - + / - Adapted From (Danks, 1995).
Analysis of metallothionein mRNA levels in the liver of these anirnals shows that
they are normal in younger tx mice and highly increased in adult tx mice (Mercer et al.,
1992). This finding suggests that metallothionein regulation is normal in these animals and
excess copper is leading to the induction of meta!lothionein. Morphological examination of
livers from adult tx mice has revealed a pattern of damage which is significantly different
from that observed in Wilson patients and bas led to some doubts as to the validity of the t.r
mouse as a model for Wilson disease (Biempica et al., 1988). However, recent studies have
mappcd the tx mutation to chromosome 8 where it has been shown to reside in the rnurine
orthologue of the human Wilson disease gene confirming that the fx mouse is, indeed, a valid
model for Wilson disease (Raucb and Wells, 1995; Reed et al., 1995; Theophilos et al.,
1996). hterestingly, the fourth metal binding domain in the mouse and rat orthologues of the
Wilson disease gene has been altered such that one or both of the conserved cysteine residues
are no longer present (Theophilos et al., 1996; Wu et al., 1994).
The LEC rat is probably the most used and hence best understood model of Wilson
disease. This inbred rat strain was established from a closed colony of normal Long-Evans
parental rats through successive generations of sibmating (Yoshida et al., 1 987). LEC rats
suffer from spontaneous hepatitis, hemolytic anemia and jaundice at approximately 4 months
of age, followed by death in 50% of animals due to fulminant hepatitis (Li et al., 1991;
Yoshida et al., 1987). The surviving 50% suffer fiom chronic hepatitis and usually go on to
develop livcr cancer (Masuda et al., 1988; Powell, 1994; Sawaki et al., 1990; Yoshida er al.,
1987). In contrast to Wilson disease, neurological abnormalities resulting from copper
accumulation in the brain are rarely seen in LEC rats. As in Wilson disease, LEC rats
accumulate copper in the liver, but semm ceruloplasmin activity is severely depressed (Li et
al., 199 1 a; Schilsky et al., I994a). Genetic analysis has shown that the mutation causing the
spontaneous hepatitis in LEC rats resides in a gene on chromosome 16 which is the rat
orthologue of thc Wilson disease gene (Muramatsu et al., 1994; Wu et al., 1994). The
mutation is predicted to give nse to a premature stop codon leading to termination of the
protein prior to the ATPase 'core'. Recently, a novel splice variant of the Wilson disease
gene in rats has been identified (Bo rjigin et a[., 1999). This variant is expressed in the brain
and eye and its activity is apparently regulated by the circadian system. Tbe functional
significance of this variant has yet to be identified.
The bedlington terrier mode1 is probably the least understood and, therefore, the least
used of the Wilson disease animal models. Copper toxicosis (CT) in bedlington temers is an
autosomal recessive disorder characterized by a defect in the biliary excretion of copper,
leading to copper accumulation in the liver and to cirrhosis (van de Sluis et al., 1999).
Despite its similarity to Wilson disease, CT in bedlington temers does have some significant
differences. Bedlington temers with CT do not show neurological abnormalities, Kayser-
Fleisher rings are absent and ceruloplasmin levels are observed to be normal (Hunt et al.,
1986; Twedt et al., 1979). Genetic analysis has failed to locate mutations in the ATP7B gene
and no linkage has been shown to esterase D (ESD) or retinoblastoma (RB), two genes which
are tightly linked to ATP7B in humans and mice (Reed et al., 1995; Scheffer et al., 1992;
Yuzbasiyan-Gurkan et al., 1993). However, acute hepatitis in the LEC rat is also not linked
to RB, suggesting that not al1 mammalian species share a syntenic relationship beween RB
and ATP7B (Sasaki et al., 1993). Taken together these results suggest that CT in bedlington
terriers may involve a gene which is not homologous to the Wilson disease gene in humans.
In support of this possibility, recent mapping studies have determined that the CT locus in
bedlington terriers maps to a region on dog chromosome 10 which is syntenic to a region on
chromosome 2 in humans, thereby elirninating ATP7B as a possible candidate gene (van de
Sluis et al., 1999). In lieu of these observations, the exact cause of CT in bedlington terriers
remains unknown.
1.6 Project Rationale
The discovery of the genes responsible for Menkes and Wilson diseases, and the
copper transporting ATPases they encode, has advanced our understanding of intracellular
copper trafficking. These two ATPases are the first members (in humans) of a rapidly
growing class of heavy metal iransporting ATPases which have recently been discovered in a
variety of organisrns. Although similar to other cation transporting P-type ATPases, the
heavy metal ATPases have several features which distinguish them from non-heavy metal
transporting ATPases (see section 1.4). Among these are the amino acid motifs (CPC and
SEHPL) which are cntical for proper fiinction of the ATPase, but are absent in the non-heavy
metal ATPases. The other unique and probably most interesting feature of the heavy metal
ATPases is the presence of a large amino-terminal domain which contains between 1 and 6
copies of the heavy metal associated (HMA) domain. The MNK and WND proteins both
contain six HMAs at their amino-terminus, while those from bacteria and lower animais
contain between 1 and 2 copies.
Comparison of the amino acid sequence from the HMAs in the MNK and WND
proteins with that from bacterial proteins clearly indicates that the human HMAs are derived
from their bacterial counterparts ( P e t W i n et al., 1994). However, one obvious difference
between the human proteins and their bacterial counterparts is the number of HMAs present.
Therefore, one of the underlying objectives of this work is to determine the significance of
having six HMAs in the human protein when only one or two seem to be sufficient in the
bacterial and yeast proteins. Several biochemical studies have hinted at the possibility that
the HMA domains may have roles in the ATPase additional to binding metals. Mutational
and functional studies have shown that the HMAs in the WND and MNK proteins are not
functionally equivalent and that not al1 are needed for the transport of copper to occur
(Forbes et al., 1999; Hung et al., 1997; Payne and Gitlin, 1998). Furthemore, in vivo
subcellular localization studies using fluorescent antibodies have s h o w that both the MNK
and WND proteins reside in the trans-golgi network and both undergo translocation to the
plasma membrane and a cytosolic vacuolar cornpartment under elevated copper
concentrations (Hung et al., 1997; Nagano et al., 1998; Petris er al., 1996). This unique
ligand-rnediated response has led to the hypothesis that the amino-terminal domain in the
iMNK and WND proteins serves as a 'sensor' for cytoplasmic concentration of copper which,
in tuni, could regulate the activity of the ATPase.
Both the HMAs and the large amino-terminal metal binding domain in the MNK and
WND proteins are novel protein entities which have not been characterized in any other
known protein. A thorough understanding of their fimction in the context of the whole
ATPase can only be gained following the characterization of the metal binding sites as well
as the structural dynamics of the amino-terminal domain as a whole. With this objective in
mind, the WCBD was systematically characterized, starting with the cloning, expression and
purification of the domain. Once purified, the WCBD was analyzed by determining i) its
metal binding characteristics, ii) its relative affinity for copper, iii) the structural effect of
metal binding, and iv) the structural properties of the copper binding sites. The results from
such an analysis may then be combined fiom fùnctional and mutational studies camied out in
systems using the entire ATPase. in the absence of a three dimensional structure, the metal
binding and structural dynamics studies will provide a wealth of information on this novel
and previously uncharactenzed domain. These studies will increase Our knowledge of the
roIe of this domain in the ATPase and could lead to the development of new pharmaceutical
agents for the treatment o f Menkes and Wilson diseases. Furthexmore, these studies will also
point the way to deciphering the structure and function o f other heavy metal hansporting
ATPases by providing a mode1 for the structure and iùnction of the large amino-terminal
metal binding domain.
CHAPTER 2
Materials and Methods
2.1 DNA Preparation
2.1.1 Ceii lines
Two separate ce11 lines were used for the maintenance of plasmid DNA and for the
expression of GST fusion proteins. E. cofi strain DHSa (supE44 AlacU169 (480 facZAM 15)
hsdR 1 7 recA 1 end4 1 w A 9 6 thi-1 relA 1) was used for the maintenance and production of
plasmid DNA for the construction of the GST expression vector constnicts, since they were
found to lead to higher purity plasmid preps (Hanahan, 1983). E. coli strain BLSl(DE3)
(hsd' gai (AcIrs857 indl Sam7 nin5 lacUVS-T7 gene 1)) were used for the expression of
GST fusion proteins as recommended in the GST Gene Fusion System Handbook
(Amersham-Pharmacia Biotech) (Studier and Moffatt, 1986).
2.1.2 Preparation of competent BLZI(DE3) and DHSa Celis
Preparation of competent BL2 l(DE3) and DH5a cells was carried out using the
calcium chloride method (Sambrook et al., 1989). Briefly, cells were grown in LB broth (1%
W/V peptone, 0.5% wlv yeast extract, 1 % w/v NaCl) to an of 0.4-0.6. The cells were
then cooled on ice for 10 minutes and harvested by centrifiigation for 10 minutes at 3000 x g,
4 ' ~ . The pellets were resuspended in ice-cold 0.1 M CaClz. The cells were then centrifûged
as above and resuspended in O. 1 M CaC12, 10% glycerol (2 ml / 50 ml original culture) and
stored at -70'~.
2.1.3 Transformation of competent BLZI(DE3) and DHSa Ce&
Calcium chloride competent cells were transformed by the heat-shock method as
described in the TA Cloning Kit (Invitrogen). Briefly, 50 pl of competent cells were thawed
on ice and mixed with 1 pl DNA @urified plasmid or tiom a ligation reaction), 2 pl 0.5 M f3-
Me and incubated on ice for 30 minutes. Following this incubation, the cells were heat-
shocked for 30 seconds in a 4 2 ' ~ waterbath and returned to ice for 2 minutes. 450 pl of
prewarmed SOC media (2% w/v peptone, 0.5% w/v yeast extract, 0.05% w/v NaCl, 20 mM
glucose, 2.5 mM KC1, 10 rnM MgC12) was added to the cells and left to recover for 1 hour at
3 7 ' ~ before they were selected on LB-ampicillin (100 mg/L) agar plates.
2.1.3.1 Preparation of glycerd stocks
A single positive clone of BL21(DE3) aansfonned with either pGEX-WCBD or
pGEX-WCBD(P) was inoculated into 5 ml LB-ampicillin and grown overnight with shaking
at 3 7 ' ~ . To 1 ml of the ovemight culture was added 0.5 ml of sterile glycerol stock solution
(65% v/v glycerol, 0.1 M MgSOs, 0.025 M Tris-HCI, pH 8.0) was added. The mixture was
split into aliquots (250 pi) and stored at - 7 0 ' ~ until needed for expression. Glycerol stocks
prepared in this manner were stable for >l year if kept at -70'~.
2.1.4 Purijication of plasmid DNA
SmaIl scale preparation of plasmid DNA was carried out using the QIAprep Minprep
Kit (QIAGEN) which uses the modified alkaline lysis method of Bimboim and Doly
(Birnboim and Doly, 1979). Transformed E. Coli were grown ovemight in 5 ml of LB broth
supplemented with ampicillin (100 mg/L). The following day, cells were hawested by
centrifugation (5 minutes, 3000 x g, 4 ' ~ ) and processed as specified in the QIAprep miniprep
handbook. Double stranded plasmid DNA was quantitated using its absorbance at 260 nm
( 1 .O unit = 50 pg/ml double stranded DNA) (Sambrook et al., 1989).
2.1.5 DNA agarose gel electrophoresis
Gel electrophoresis was used to determine the size and purity of purïfied plasmids
and for the separation of different-sized DNA fragments pior to purification. Agarose slab
gels using the TAE buffer system were used (Sambrook et al., 1989). Al1 1% agarose gels
were prepared in 0.5X TAE buffer containing 1 pg/ml ethidium bromide and were run at a
constant voltage (100 V) until adequate separation was achieved. Ethidium bromide stained
nudeic acid bands were visuaiized using ultraviolet iight.
2.1.6 Purijication of DNA restriction fragments from agarose gels
DNA hgments were separated by agarose gel electrophoresis and purified using the
SephaglasTM BandPrep Kit (Amersham-Pharmacia Biotech), which is a modification of the
Na1 method (Vogelstein and Gillespie, 1979). Purified DNA fragments were quantified
using their absorbance at 260 nm and used without further purification in subsequent
procedures.
2.1.7 DNA Sequencing
Double-stranded plasmid DNA was sequenced by the dideoxy sequencing method
(Sanger et al., 1977) using the T7 SequencingTM Kit (Amersham-Pharmacia Biotech)
according to the manufacturer's directions. The purity of the plasmid preparations was
checked prior to sequencing by measuring the OD (260 nm 1 280 nm) ratio and ensuring that
it was at least 1.8.
2.1.8 Restriction envme digests and ligation reactiuns
Restriction and modiQing enzymes used in these reactions were obtained from
Amersham-Pharmacia Biotech and New England Biolabs (NEB). The reactions were carried
out under the conditions specified by the manufacturer, using the buffers supplied. Al1
Iigation reactions were carried out using T4 DNA ligase fiom NEB. The vector and insert
were ligated at a 1 : 1, 1 :3, 1 5 , and 1 : 10 molar ratio starting with 50 ng of vector and the
corresponding amount of insert calculated using equation 1.
(Y bp insert)(SO ng vector) X ng insert =
(size in bp of vector)
The value obtained for X fiom this equation gives the amount of insert for a 1 : 1 molar ratio.
Multiples of X (3X, 5X and 10X) were used for the other ratios. Generally 400 units of
ligase (NEB unit definition) were added to each Iigation reaction, total reaction volume was
20 pl. Ligation reactions behveen fragments of DNA with compatible ends were carried out
ovemight at 16 '~ . Usually, 1 pl of the ligase reaction was used to tmnsform bacteria and the
remainder was stored at -20'~.
2.2 Construction of Expression Vectors
2.2.1 Construction o f p GEX- WCBD
The 2-kilobase cDNA encoding the Wilson disease protein copper binding domain
(WCBD), was produced in two fragments from total human liver RNA using reverse
transcriptase-polymerase chain reaction (RT-PCR). This reaction was performed by J.R.
Forbes in the laboratory of Dr. D.W. Cox. Primers were designed to create a BamHI
restriction site at the 5' end and a Sul 1 site at the 3' end. The two cDNA fragments
overlapped at an Nsil restriction site. The pnmers used for the 5' fragment were (5' side) 5'-
TATCGGATCCATGCCTGAGC-AGGAGAGA-3' (5'-BamHI), and (3' side) 5' -
AACTTTAAAATTCCCAGGTGG-3' (OL- 1); 3' fragment (3' side) 5'-
ACTTGTCGACTCACTGCTTTATTTCCATTTTG-3' (3'-Sa1 1), and (5' side) 5'-
GGAATGCATTGTAAGTCTTGGCG-3' (OL-2). A stop codon (bold) was inserted just
before the San site; restriction sites are underlined. The two fragments were digested with
BarnHI and .Msi 1 or Sul 1 and Nsi 1, respectively, and following purification fkom an agarose
gel, ligated into the BamHi and Sul 1 sites of the GST expression vector pGEX-4-T-2
(Amersham-Pharmacia Biotech, Inc) to create the expression vector pGEX-WCBD (Fig.
2.1). This vector contains a thrombin cleavage site between the GST moiety and the WCBD.
The fidelity of the constmct was confinned by dideoxy sequencing.
2.2.2 Construction of pGEX- WCBD(P)
The WCBD DNA insert was excised from pGEX-WCBD using BamHI and Sali,
purified on a 1% agarose gel and inserted into the BamHI and Sul 1 sites of the GST fusion
expression vector pGEX-6P-2 (Amersham-Pharmacia Biotech, Inc) to create the expression
vector pGEX-WCBD(P) (Fig. 2.2). This vector contains a PreScissionTM (human rhinovirus
3C protease) cleavage site between the GST moiety and the WCBD (Walker et al., 1994).
The fidelity of the construct was confirmed by dideoxy sequencing.
C Leu - Val - Pro - Arg - J - Gly - Ser
Figure 2.1. Parental and Modifxed GST Fusion Expression Vectors. A. parental GST fusion expression vector, B GST-WCBD expression vector containing - 2 Kb WCBD cDNA insert, C thrombin protease recognition site located between GST and WCBD. Cleavage by the protease occws at the Arg - Gly bond. D thrombin 5 and 3 amino acid consensus sequences. P 1 and P2 are hydrophobie amino acids, P3 and P4 are nonacidic amino acids, P 1' or P2' are Gly
B Leu-Glu-Val-Leu-Phe-Gln- J -GIy-Pro
Figure 2.2. GST- WCBD(P) Expression Vecfor with PreScissionTM Cleavage S i t e . A GST-WCBD(P) expression vector with PreScissionm protease ckavage site between GST and WCBD. B PreScissionm protease recognition site. Cleavage occurs at the Gin - Gly peptide bond.
2.3 Expression and Purification of GST-WCBD and GST-WCBD(P)
2.3.1 Bu ffer and reagent preparuîion
Al1 buffers and reagent solutions were prepared using purified distilled water with a
conductivity of at least 18 M. Deionized urea was used in al1 buffers requiring it and was
prepared as follows: approxirnately 100 - 150 g of AG501-X8 (D) mixed bed resin @IO-
RAD) was added to 2 L of 8 M urea in distiIled water and stirred overnight at RT; the resin
was then removed from the solution by vacuum filtration. Al1 buffers not containing
reducing agents (DTT or f3-Me) were argon purged for at least 30 minutes at RT prior to use.
Vinyl, powder free gloves (Baxter Healthcare Corp) and clear plastic pipette tips were used
in al1 manipulations to avoid metal contamination. Residual metal contamination in dialysis
tubing was removed prior to use by boiling the tubing for approximately 1 to 2 hours in 5 to
6 changes of distilled water.
2.3.2 Expression of GST- WCBD and GST- WCBD(P)
An aliquot of a glycerol stock containing BL2 1 (DE3) transformed with either pGEX-
WCBD or pGEX-WCBD(P) was inoculated into 100 ml of LB supplernented with ampicillin
(100 pg/ml) and grown ovemight at 3 7 ' ~ with shaking. Following overnight growth, the
culture was divided in two and diluted into 2 x 1.5 L fiesh LB supplemented with ampicillin
and 100 PM CuS04 (Tris-buffered, pH 7.0) in 2.8 L Fembach flasks. The cultures were
grown with shaking at 3 7 ' ~ until midlog phase was reached (kW of 0.6-0.8), at which point
they were induced with 0.1 mM IPTG for 3-4 hotus. Following the induction period, the
cells were hawested by centrifugation at 4420 x g for 15 minutes, at 4 ' ~ . Tbe supernatant
was poured off and the bacterial pellets were either stored at -20 '~ ovemight or lysed
immediately.
2.3.3 Purification of GST- WCBD and GST- WCBD(P)
The bacterial pellets were resuspended in lysis buffer (20 mM Tris-HCI, pH 8.0, 130
mM NaCl, 1 mM EDTA, 5 mM DTT, 1 0% glycerol, I mM PMSF, 1 % Triton X- 1 00 and 0.1
mg/ml lysozyme) and lyseci by two cycles of fieeze-thaw. The lysate was centrifüged at
118,000 x g for 45 minutes, at 4 ' ~ . The soluble fraction was decanted and stored at 4 ' ~ .
Fusion protein which localized to inclusion bodies was solubilized and refolded, essentially
as described previously (Dombroski et al., 1992). Briefly, inclusion bodies were solubilized
by homogenization in solubilization buffer (50 mM Tris-HCI, pH 8.0,6 M Urea, 8 mM DTT,
1 mM EDTA) and the reviilting suspension was centrifûged at 1 18,000 x g for 45 minutes at
4 ' ~ . Solubilized fusion protein was refolded by extensive dialysis against refolding buffer
(50 mM Tris-HC1, pH 8.0, 20% glycerol, 5 mM DTT, 1 rnM EDTA). Refolded fusion
protein was combined with the soluble fraction. The resulting mixture was made 1% in
Triton X- 100 and applied to a glutathione Sepbarose 4B column previously equilibrated with
TEND-T (20 mM tris-HC1 pH 8.0, 130 mM NaCl, 1 m M EDTA, 5 mM DTT, 1% Triton X-
100). The column was washed extensively with TEND-T followed by TEND (same as
TEND-T but without Triton X-100). The fusion protein was eluted fiom the column with
TEND-U (20 rnM Tris-HCI, pH 8.0, 130 NaCl, 1mM EDTA, 5 mM DTT, 6 M Urea).
Protein elution was monitored by OD2so readings and samples fiom each fraction were
analyzed by SDS-PAGE.
Fractions containing fusion protein were combined and applied to a Q-sepharose fast
flow column (anion exchange), previously equilibrated with TEND-U at a flow rate of 2.2 ml
/ minute, collecting 5 ml fractions. After sample application, the column was washed
extensively with TEND-U and ODzao reading were taken on each fraction to monitor protein
content. When readings retmed to baseline values, the fbsion protein was eluted with
a linear salt gradient (130 - 250 mM NaCl). Peak fractions were analyzed by SDS-PAGE
and those containing fùsion protein were pooled and refolded as described above.
2.3.4 SDS-PA GE
SDS-PAGE was performed on a mini slab gel apparatus (Hoefer Scientific
Instruments) in the buffer system developed by Laemrnli (Laemmli, 1970). To visualize the
fusion protein as well as the WCBD, we prepared 10% separating gels overlaid with 4%
stacking gels. The reduction of disulfide bonds and the incorporation of SDS were achieved
by mixing protein samples with an equal volume of 2X loading buffer containing 0.72 M p-
Me, boiling for 5 minutes, and immediately loading onto the gel. Gels were run at a constant
voltage (175 V) until separation was achieved, as determined fiom the mobility of the dye
front. Following electrophoresis, the gel was stained in a 0.125% w/v solution of Coomassie
Brilliant Blue R-250 dissolved in 40% v/v methanol, 10% v/v acetic acid, 50% distiiled
water, with sbaking for 15 minutes at RT. The gel was destained by incubation in 10% v/v
methanol, 10% v/v acetic acid, 80% distilled water, with shaking overnight. Destained gels
were photographed using the UVP gel documentation system in conjunction with UVP-Grab-
I F " software version 1.57b (DiaMed Lab Supplies). Molecular weights of the proteins were
estimated by comparison with wide range molecutar weight marker run in paralle1 (200 - 6
kDa, NOVEX Mark 12 Standards).
2.3.5 Protein quanriration
Quantitation of the amount of fiision protein or WCBD was camed out using the
BCA Protein Assay Kit (Pierce). The assay was carried out as outlined in the instruction
manual for the standard protocol. The concentration of the protein sample was detennined
by comparison to a standard curve denved tiom BSA (O - 50 pg, run in duplicate). Results
fiom this assay agreed very closely to those obtained by amino acid analysis.
2.3.6 N- Terminal Amino Acid Sequencing
N-terminal amino acid sequencing was carried out on the purified WCBD to confirm
its identity. The samples were run on 10% SDS-PAGE gels and electroblotted ont0 PVDF
membranes (BIORAD). Electroblotting was carried out in a Hoefer electroblot transfer
apparatus (Arnersham-Phannacia Biotech) at a constant current of 0.5 A for 45 minutes using
CAPS transfer buffer (10 mM CAPS pH 1 1 .O, 10% v/v methanol) cooled with circulating
water. Following blotting, the membrane was washed in two changes of transfer buffer and
then stained with O. 1% w/v Coomassie Bnlliant Blue R-250 dissolved in 50% v/v methanol,
50% distilled water for 1-2 minutes. The membrane was then destained with 50% v/v
methanol, 10% v/v acetic acid, 40% distilled water for 5 minutes and then washed with
several changes of distilled water for 5-10 minutes. The membrane was air dned and
submitted for N-terminal sequence analysis. Seven cycles of automated amino acid
sequencing was carried out on a Porton instruments Gas-Phase Micro Sequencer mode1 PI
2090E with online PTH amino acid aoalysis using a Hewlett-Packard HPLC, mode1 1090L.
N-terminal sequencing reactions were carrïed out using Edman degradation (Matsudaira,
1989)
2.3.7 Amino Acid Analysis
Purified fùsion protein was analyzed for total amino acid content to confirm the
identity of the protein and to determine concentration. The fusion protein was hydrolyzed
with hydrochloric acid (6 M) for 48 hours and the amino acids were reacted with
phenylisothiocyanate (PITC) to form phenylthiohydantoin (PTH) amino acid denvatives.
The PTH-amino acid derivatives were analyzed and quantitated by HPLC in cornparison with
a standard mixture of PTH-derivitized amino acids (Smith, 1997). Fusion protein
concentration was determined by dividing the co~centrations of PTH-Leu, PTH-Ala, PTH-Ile
by 79, 66, and 59 respectively (corresponding to the molar amounts of each residue present
in the fusion protein) and calculating the average of the values obtained.
2.4 Removal of GST from GST-WCBD and GST-WCBD(P)
2.4.1 Thrombin cieavage of GST- WCBD
Thrombin cIeavage of GST-WCBD was either carried out on the cotumn or on the
purified hsion protein in solution. For cleavage of the column-bound fusion protein, 10
units of human thrombin (Sigma-Aldrich) / mg of fusion protein was added to 1 bed volume
of TEND (20 m M Tris-HCI, pH 8.0, 130 mM NaCl, 1 mM EDTA, 5 mM DTT) and applied
to the column. The reaction mixture was incubated at RT for 30-45, minutes followed by
elution of pure WCBD in the same buffer. Thrombin was inhibited by the addition of PMSF
to a final concentration of 1 mM. For cleavage of purified fision protein in solution, 10 units
of human thrombin / mg of fusion protein was added to the protein solution and the reaction
was incubated for 30-45 minutes. After the incubation period, thrombin activity was
inhibited by the addition of PMSF (1 rnM final concentration) and the mixture was applied to
a glutathione affinity column. The flow-through which contained the purified WCBD was
collected.
2.4.1.1 Optimizaîion of thrombin cleavage cortditions
Thrombin cleavage conditions for GST-WCBD were optimized in the following
marner to minimize the non-specific degradation of the WCBD. To a 2 ml solution of the
purified fusion protein (0.48 mg / ml) were added 9.5 cleavage units of human thrombin and
the reaction mixture was incubated at RT. Samples (50 pl) were taken out every 30 minutes
for the first 4 hours and then every hour thereafter and mixed with an equal volume of 2X
loading buffer containing 0.5 mM PMSF. The samples were boiled for 5 minutes and
analyzed on 15% SDS-PAGE slab gels.
2.4.2 PreScission- clerrvage of GST- WCBD(P)
Removal of GST fiom GST-WCBD(P) was carried out on the purified fiision protein
in refolding buffer (50 mM Tris-HCI, pH 8.0, 1 mM DTT, 20% glycerol). Briefly, 1 unit of
PreScissionm protease (Amersham-Pharrnacia Biotech) / mg fusion protein was added to the
protein solution and the reaction mixture was incubated at SOC for 16-48 hrs. The progress of
the cleavage reaçtion was monitored by removing aliquots several time intervals and
analyzing them by SDS-PAGE. Once the reaction was judged complete, the mixture was
passed over a glutathione affinity column to remove free GST and the protease (the protease
is supplied as a h i o n with GST). The flow-through contained the purified WCBD.
2.5 Characterization of the Metal Binding Properties of WCBD
2.5.1 Imnrobifized Metal Ion A ffiniw Chromatography (IMA C)
Samples of the purified fusion protein were dialyzed against MAC buffer (20 mM
NaH2P04, pH 7.0, 0.5 M NaCl) with or without 6 M urea and applied to chetating sepharose
fast flow columns (Amersham-Pharmacia Biotech) charged with either Co(ll), Ni(II), Zn(II),
Cu(II), Fe(II) or Fe(III) as per the manufacturer's instructions. Elution of the hsion protein
from the matrix was achieved either by lowering the pH to 6.0 or 4.0 or by the sequential
addition of chelators (EDTA, imidazole, BCS). Samples of each fraction were analyzed on
10% SDS-PAGE gels.
2.5.2 6 5 ~ r 1 ( ~ Blotting Assay
65 Zn(Q blotting analysis was performed essentially as described previously (Schiff et
al., 1988) with the following modifications. Samples of GST-WCBD, WCBD, GST and
estrogen receptor DNA binding domain (ERDBD) were subjected to SDS-PAGE and
electroblotted onto nitrocellulose membranes (BIORAD). Electroblotting was carried out in a
Hoefer electroblot transfer apparatus (Amersham-Pharmacia Biotech) at a constant current of
0.3 A for I .5 hours using CAPS transfer buffer (10 mM CAPS, pH 1 1.0) cooled with
circulating water. Following electroblotting, the membranes were equilibrated in metal
binding buffer (100 rnM Tris-HC1, pH 7.0, 50 mM NaCl, 1 rnM DIT) for 2 hours at RT with
shaking. The membranes were then probed with 10 pCi of ' 'z~cI~ (30 pM final Znfl1)
concentration) in 20 ml metal binding buffer, without DTT unless otherwise noted, for 1 hour
at RT, with shaking. The membranes were then washed twice (15 minutes each) with the
same buffer. The membranes were then wrapped in plastic wrap and exposed to Biomax MR
film (Kodak) for 22 hours at -70'~ with intensifying screen. Following autoradiography, the
film was developed using a M3SA X-OMAT processor (Kodak).
2 S.2.l Eflect of pH on 6 s ~ n ( ; r ~ Bloning Assay
The effect of pH on the binding of zinc to the domain was assessed using the 6S~n(lI)
blotting assay. The assay was c d e d out as described above, except that the pH of the metal
binding buffer was varied from 6.5 to 9.0 in steps of 0.5 pH units (6 separate blots). The
experiment was carried out using GST-WCBD, WCBD, GST and ERDBD.
65 2.5.2.2 Eflect of DTT on Zn(Z1) Blotting Assay
The effect of DTT on the binding of zinc to the domain was assessed using the
65 Zn@) blotting assay. The assay was carried out as described above, at either pH 8.0 or pH
6.5, under the following conditions: in the presence of 1 m . DTT during the addition of
metal, in the complete absence of DTT throughout the assay; and in the presence of 1 mM
DTT only dunng the equilibration step (standard assay conditions). The experiment was
carried out using GST-WCBD, WCBD, GST and ERDBD.
2.5.3 Contpeiition " ~ n f l l ) Blofting Assay
The standard 6 s ~ n ( ~ ~ ) blotting assay was cm-ied out as described above, using
purified WCBD, except that the membranes were probed with 6 S ~ n ( ~ in the presence of a
non-radioactive competitor metal at concentrations ranging fkom 0.01 mM to 1 mM. Blots
probed with 6s~n(tI) in the absence of a competitor metal were used as controls. The sigoals
from the autoradiograms were quantified using the gel-plotting macros in the NIH Image
1.61 software package. The percent zinc bound was calculated by dividing the signal
obtained in the presence of a competitor to that obtained in its absence (taken as 100% zinc
binding). In some instances the strips were stained with O. 1% wlv Amido Black dissolved in
45% vlv rnethanol, 10% v/v acetic acid, 45% distilled water for 1-2 minutes, followed by
destaining in several changes of 45% vlv methanol, 10% vlv acetic acid, 45% distilled water.
Stained membranes were analyzed to confirm that equal amounts of protein were present in
each lane.
2 S.4 Stoichiometry of metal binding
The copper binding capacity of the domain was evaluated by NAA. Briefly, urea-
denatured fusion protein was dialyzed against refolding buffer containing copper (20 mM
Tris-HC1, pH 8.0, 10% glycerol, 0.1 mM DTT, 0.1 mM CuC12). Following dialysis, the
protein concentration was detennined using the BCA protein assay (Pierce), and the samples
were then dialyzed extensively against 1 % fonnic acid (pH 3.0) and Iyophilized over a 4 day
period on a Freezernobile 25XL lyophilizer (Virtis). Approximately 1.225 mg of the
lyophilized fusion protein (0.0129 prnol) was submitted for copper determination by NAA.
2.5.5 Neutron Activation Analysis (i'VA4)
Neutron activation analysis was performed at the Slowpoke reactor facility at the
University of Toronto. In general samples analyzed for copper were irradiated for 2 hours
using a neutron flux of 1 .O x 10" neutrons/cm2/s. Samples were counted after a 3 hour delay
for 600 seconds per sample. The amount of copper is each sample was determined by
comparing the amount of (î+ = 5.1 minutes, 1039 keV) in the sample to an external
standard using the comparator method. Samples analyzed for copper and zinc were
irradiated for 16 hours using a neutron flux of 2.5 x 10" neutrons/cm2/s. Samples were
counted afier a 6 hour delay for 1000 seconds per sample. The amount of copper and zinc in
each sample was determined by comparing the amount of %u and 6 9 m ~ n (L, = 12.8 hours,
5 1 1 keV and t;, = 13.8 hours, 438.7 keV respectively) in the sample to an extemal standard
using the comparator method (Hancock, 1976; Hancock, 1984; Hancock, 1978).
2.6 Characterizatioa of the Copper Binding Sites
2.6.1 Preparation of apo-pro rein
Meta1 removal fiom the fùsion protein as well as the WCBD was achieved as follows:
A solution containing the fusion protein or the WCBD was made 0.5 M in p-Me and
incubated at 4 ' ~ for 6-8 hrs. Following this incubation, the proteins were rapidly
precipitated by the addition of TCA to a final concentration of 10%, followed by
centrifugation at 3000 x g for 15 minutes at 4 ' ~ . The protein pellet was resolubilized in 0.5
M Tris base, 6 M Urea, 0.5 M fi-Me with sbaking at 4 ' ~ ovemight. AAer resolubilization,
the protein was again precipitated with TCA and resolubilized in 0.5 M Tris, 6 M Urea. The
protein was then refolded by dialysis against modified refoiding buffer (50 mM Tris-Acetate,
pH 8.0,20% glycerol) and then refolding buffer \vithout glycerol. The protein concentration
was deterrnined by the BCA protein assay (Pierce) and then analyzed for metal content by
NAA.
2.6.2 Preparation of Sampfes For XAS and CD anaiysis
Fusion protein and WCBD containing V ~ ~ O U S amounts of copper or zinc were
prepared as follows. DTT was added to samples of apo-GST-WCBD or apo-WCBD to a
final concentration of 1 mM and incubated on ice for 30 minutes. The required amount of
copper or zinc (using CuSOa and ZnSOs) was then added to achieve the desired final molar
ratio of metaVprotein and incubated at RT for at least 30 minutes. A 15 fold molar excess of
metal was usually used to achieve full reconstitution of the protein with metal. The unbound
metal and DTT were then removed by extensive dialysis against 25 mM Tris-Acetate pH 8.0
(25 rnM Tris-HCI pH 8.0 for CD samples). The protein concentration was again conf~rmed
by the BCA protein assay and metal content was assessed by neutron activation. Al1 dialysis
buffers were purged with argon before use and diaIysis was performed in sealed containers.
Typical concentrations of apo-fusion protein used in this procedure were between 1 and 5
mg/ml. In some cases an alternative procedure for the addition of copper or zinc to the apo-
protein was used to eliminate the possible interference of exogenous sulfûr-containing
compounds. In this method, the protein sulhydryls were reduced by the addition of a 1:1
molar ratio (with respect to cysteine concentration) of TCEP. Copper was then added to the
apo-protein in the presence of 1 mM ascorbic acid. Ascorbic acid was omitted during the
addition of zinc to the apo-protein.
2.6.2.1 Preparation of & X4S Sampies
Dry XAS samples were prepared as described above (2.6.2), except that the samples
were dialyzed against 25 m M Ammonium Acetate, pH 7.5 afier incubation with the metal.
Following protein concentration and metal content determination, the samples were
lyophilized by fust flash fieezing them using a dry ice / acetone bath and then lyophilizing
them for 4 days on a Freezemobile 25XL lyophilizer (Virtis). After lyophilization, the
samples were shipped to Dr. Que's lab at the University of Minnesota for XAS analysis.
2.6.2.2 Preparation of Liquid XAS and EPR Samp /es
Liquid samples for XAS and EPR were prepared as described above (2.6.2), except
that the apo protein solutions were more concentrated (16 mg/ml) and, after incubation with
metal the protein was dialyzed against 35 mM Tris-Acetate, pH 8.0, 30% glycerol. Empty
EPR tubes and XAS sample holders were obtained fiom Dr. Que's lab and filled with 0.75 ml
and 0.2-0.3 ml of sample, respectively. The XAS sample was fiozen by placing the holder
on top of a piece of dry ice while the EPR sample was flash fkozen using liquid nitrogen.
The filled tubes and sarnple holder were then shipped back to Dr. Que's lab on dry ice for
analysis.
2.6.3 EX4 FS and XANES AnalysrS of Cu-GST- WCBD
EXAFS and XANES analysis were carried out by Hua-Fen Hsu in Dr. Que's Iab, as
follows. X-ray absorption spectra were collected between 8799 eV and 9726 eV at beamline
X9B of the National Synchrotroa Light Source (NSLS) at Brookhaven National Laboratory.
The protein data were collected in fluorescence mode (Aexp (CflCo)) at 10-20 K by using a
flat Si(220) double-crystal monochromator and a 13-element Ge detector. The
monochromator was calibrated using the edge energy of copper foi1 at 8979.0 eV. Data
extraction and analysis was perfonned as previously described (Scarrow et al., 1987; Shu et
al., 1995). To fit the EXAFS contribution from copper scatterers, the empirical amplitude
reduction factor A and the shell-specific edge AE were extracted from the XAS data of
crystallogaphically characterized mode1 complexes, [ C U ( S C ~ H ~ ) ~ ] ~ - and [~u(Im)4]~+.
2.6.4 Oxidutive releese of copper from WCBD
To samples of copper reconstituted fusion protein (1 -8 mg/ml, 18.4 ,A) were added
either hydrogen peroxide (HIOZ) or cent ammonium nitrate (Ce(NH&(N03)6) to a final
concentration of 250 pM and the reaction was incubated at RT for 2 hours. One sample did
not receive any oxidizing agents and was used as the controt. The samples were then
dialyzed extensively against 25 m M Tris-Acetate, pH 8.0, at 4 ' ~ and an aliquot fiom each
sarnple was taken (1 -53 mg protein) and submitted for copper determination by NAA.
2.7 Conformational Analysis of GST-WCBD and WCBD
2.7.1 Anaiysis of protein structural changes upon metaf binding
Samples for CD analysis containhg either copper or zinc were prepared as described
in section 2.6.2 and analyzed on a Jasco 5-720 spectropolarimeter. For analysis of changes in
secondary structure, samples were loaded into a O. 1 cm path length CD ce11 and spectra were
recorded from 300 - 190 nm. For analysis of changes in tertiary structure, samples were
loaded into a 2 cm path length CD ceil and spectra recorded from 400 - 250 m. In botb
cases, spectra were recorded using the parameters listed in table 2.1. The spectra were
Table 2.1 CD Parameters for the ConformationaI
GST-WCBD and WCBD
- .
Resolution 1 0.2 n m l
Band Width Sensitivity
1.0 nm 10 mdeg
1 Accumulation 1 4 1
Response Speed
1 second 50 d s e c o n d
corrected for the contribution of the buffer and noise reduced and the data were converted to
molar ellipticity using the software supplied with the spectrometer. Molar ellipticity values
per residue were calculated by dividing the molar ellipticity by either 867 for GST-WCBD or
649 for the WCBD.
2.7.2 Estimation of secondaty structure content
Secondary structure content estimation of both the WCBD and the fusion protein
reconstituted with various amotmts of copper and zinc was carried out using the SELCON
(self consistent method) and K2D (neural network) programs (Andrade et al., 1993; Bohm et
al., 1992; Merelo et al., 1994; Sreerama and Woody, 1994; Sreerama and Woody, 1993).
For the SELCON program, CD data between 260 - 200 nm and the secondary structure
assignrnents and protein data base of W. C. Johnson Jr., containing 33 proteins, were used
(Johnson, 1990). Parameters for the SELCON programs were as follows:
CONSTl (limit for sum of fiactions /sum fk - 1.01) CONST2 (limit for hctions, fk >)
For the K2D program, CD data between 240 and 200 nm was used. The SELCON program
was used to predict a-helix, fi-sheet, 6-turns, and other conformations, while the K2D
0.15 -0.15
NITR (max number of iterations for self-consistency) CONVRG (convergence criteria for sel-consistency)
program was used to calculate a-helix, fi-sheet and random conformations. The KîD
10 0.005
prograrn was found to give very good estimates of f3 structure and this value was used in
conjunction with the values from SELCON to estimate overall protein secondary structure
content (Greenfield, 1996). These and other programs for the estimation of secondary
structure from CD spectra are available a t the following website
http://www2.urndnj.edulcdrwjweb/index.htm. 1 gratefiilly acknowledge the assistance of Dr.
Norma J. Greenfield in the use and application of these programs.
CHAPTER 3
Metal Binding Properties of the N-Terminal Copper Binding Domain from the Wilson Disease Copper
Transporting P-Type ATPase (ATP7B)
The rnajority of the material presented in chapter 3 has been published in: DiDonato, M.,
Narindrasorasak, S., Forbes, J.R., Cox, D.W., and Sarkar, B. ( 1 997) Expressiori, Purification,
and Meta1 Binding Properties of the N-terminal Domain fiom the Wilson Disease Putative
Copper-transporting ATPase (ATP7B). J. BioZ. Chem. 272(52), 33279-33282.
3.1 Introduction
In many ways the presence of copper in biological systems is a double edged sword.
Although trace amounts of copper are absolutely required to sustain life, an excess or a
deficiency of copper can lead to serious biochemical defects (Danks, 1995; DiDonato and
Sarkar, 1997). Many aspects of copper transport bave been studied in the past, however in
recent years, key discoveries have been made which have allowed us to start dissecting the
detailed mechanism of intracellular copper traficking and copper homeostasis. The greatest
of these discoveries has been the isolation of the gene responsible for both Menkes (Chelly et
al., 1993; Mercer et al., 1993; Vulpe et al., 1993) and Wilson (Bull et al., 1993; Tanzi et al.,
1993) diseases, two major genetic disorders of copper metabolism in humans. The discovery
of these genes and the putative copper transporting P-type ATPases they encode, have
opened up new avenues of research in the copper transport are.1.
Understanding the mechanism of action of these proteins in vivo will be a necessary
first step in understanding the disease mechanism. ïhese ATPases represent a new class of
human heaw metai traasporting ATPases and have several interesting features. Although
they share a great deal of homology with each other as well as with other cation transporting
ATPases, they do have some features which are not present in other ATPases (Bull et al.,
1993; Tanzi et al., 1993; Vulpe et al., 1993). Among these are a conserved Cys-Pro-Cys
motif in transmembrane six, a conserved Ser-Glu-His-Pro-Leu motif in a cytoplasmic loop of
unknown fünction and a large amino-terminal region containing six copies of the heavy
rnetal-associated domain (HMA). The consensus sequence for the HMA, which spans the
complete domain is: &IVN]-x(2)-[LIVMFA]-x-C-x-[STAGCDNH]-CIx(3)-FIVFG]-x(3)-
[LIVI-x(9,11)-DVA]-x-[LVFYS]. The HMA has also been found in the metal binding
domains of various bacterial heavy metal transporting proteins (Odermatt et al., 1994; Phung
et al., 1993). The two cysteine residues (bold) are strictly conserved and are most likely
involved in metal ligation.
The presence of the large amino-terminal copper binding domain in the Menkes and
Wilson proteins raises many questions as to its fùnction. The repeated copper-binding motif
represents a totally new and uncharacterized protein and its copper-binding site. Although it
is likely that the rwo conserved cysteine residues in the motif participate in the ligation of
copper, nothing m e r is known about this motif or its function in vivo. To begin to
characterize this motif and the copper bindiag sites, we have developed a bacterial expression
system for the production of the entire amino-terminal metal binding domain of the Wilson
disease protein (ATP7B) îüsed to glutathione-S-emsferase (GST).
3.2 Results
3.2. f Expression and purljica fion of GST- WCBD, GST- WCBD (P), and WCBD
The 2 kbp cDNA encoding the entire amino-terminal metal binding domain (residues
1-649, -70 kDa in size) of the Wilson disease ATPase was produced, cloned and expressed
as described in sections 2.2 and 2.3. As iilustrated in figure 3.1, E. c d i transformed with the
expression plasmid pGEX-WCBD exhibited slightly better growth following induction in
media supplemented with increasing amounts of copper when compared to E. coli
transformed with the parental expression vector pGEX-4T-2. This indicates that the
expressed fusion protein may be offsetting the damaging effects of excess copper on the
bacteria by binding it and lowering its fiee concentration.
A typical purification of WCBD, GST-WCBD, and GST-WCBD(P) is shown in
figure 3.2. Approximately 70% of the expressed fusion protein was found in inclusion
bodies, while 30% remained soluble. Fusion protein localized to inclusion bodies was
recovered by solubilization and refolding from urea. Using a combination of glutathione
affinity chromatography, and denaturing ion exchange chromatography the fùsion protein
was obtained in a >90% pure form. The identity of the protein was confinned by amino-
terminal sequencing.
Treatment o f the purified fiision protein eluted fiom the glutathione affinity column
with 6 M urea in the absence of reducing agents witb BCS, gave rise to a reddish-orange
color (hm, = 480 nm) indicative of the CU(I)(BCS)~' complex (Chen et al., 1996). This
suggests that the domain binds copper in the +1 oxidation state and that copper is being
incorporated into the protein during its production in bacteria. Absorption spectra of the
purified fusion protein in the visible and UV regions do not show any features which would
O 1 2 3 4 Time (hm)
Figure 3.1 Ejfect of Copper on BLZZ(DE3) Growth Following Induction. Growth of BL2 l(DE3) traasformed with either A pGEX- 4T-2 or B pGEX-WCBD. Tris-buffered copper was added to the media at the indicated finai concentration at time of induction. At the times indicated, O&()() readings were taken on aliquots of cells, diluted 10 fold.
, GST- WCBD
W C B D
Figure 3.2 Sztmmary of Typical Purr@cation of WCBD and GST- WCBD. GST-WCBD was expressed and purified as described in section 2.3. A. purified WCBD eluted fiom glutathione-sepharose 4 8 resin following 45 minutes cleavage of GST-WCBD with thrombin on the colurnn. B. GST-WCBD eluted with urea fiom glutathione affinity resin following solubilization and refolding fkom inclusion bodies. C. purified GST-WCBD eluted from Q-sepharose FF anion exchange resin. Proteins derived from the modified vector pGEX-WCBD(P) were expressed and purified using the sarne procedure except, cleavage was camied out using the PreScission protease.
indicate the presence of copper in the +2 state, M e r suggesting the presence of CuO. The
metal bound to the domain appears to be fairly strong since it is able to withstand denaturing,
refolding and the presence of reducing agents (10 mM glutathione, 5 rnM DTT). Metal
binding under these conditions has also been observed for the estrogen receptor DNA
binding domain (2 zinc finger protein having 4 cysteines in each finger) where a combination
of low pH, urea, DTT and chelating agents are needed to remove the bound metal (Conte et
al., 1996).
3.2.2 Metal binding characteristics of GST-WCBD and WCBD
3.2.2.1 Immübifited metai ion affrniry chromptograpiy (7MA C)
To investigate the metal binding properties of the domain, both immobilized metal
ion affinity chromatography (MAC) and 6 5 ~ i n c binding assays were carried out using the
fusion protein and the cleaved domain. The results of IMAC using the purïfied fusion
protein are presented in figure 3.3. The expenment was run both under non-denaturing and
denaturing conditions in the absence of reducing agents with nearly identical results. GST
alone did not bind to uncharged metai columns and had sorne weak, non-specific interactions
under non-denaturing conditions on metal charged columns. The fusion protein did not bind
to columns charged with either Fe@) or Fe(II1). The other transition metals showed varying
affinities for the domain. Based on the sequential elution conditions used, the apparent order
of affinity for the different transition metals for the domain was:
Cu(Ii)>>Zn(II)>Ni(n)>Co(II).
The variation in affinity may reflect the inability of the protein metal binding sites to
adopt the preferred conformation for that metal atom. Interestingly, the fusion protein could
PH Imidazo le 50 rnM , y EDTA 50rnM BCS
Figure 3.3 Immobilized Metul Ion Affinity Chrornaiography (IMAC) of GST- WCBD. Samples (S) of the fusion protein were applied to chelating sepharose FF columns charged with the indicated metal under non-denaturing conditions. Protein which did not bind to the column came off in the flow through (FT). Bound protein was eluted by sequential treatment with low pH (6.5 - 4.0), increasing amounts of imidazole (LOO - 500 mM), EDTA and BCS. No binding was observed to columns charged with i o n (+2 or +3). Very similar results were obtained using denaturing conditions.
only be released from the Cu(1I)-charged column by the addition of the specific copper
chelator BCS. Also, elution of the fusion protein with BCS was accompanied by the
formation of the reddish-orange CUO(BCS)~- cornplex, indicating that copper was bound to
the domain in the + l oxidation state. Considering that the colwnn was charged with Cu(I1)
in the absence of reducing agents, this observation suggests that Cu@) atoms may be
reduced to Cu(T) upon binding to the domain.
3.2.2.2 Neutron activation anaiysis (nAA)
To determine the stoichiometry of metal binding to the domain, neutron activation
analysis was used to quantitate the amount of copper bound to GST-WCBD following
reconstitution with excess rnetal during refolding. A sarnple (1.225 mg) of GST-WCBD
(-96 kDa, 0.0 129 pmol) was analyzed by NAA and found to contain 0.089 I 0.005 pmol of
copper. This analysis revealed that 6.5 - 7.3 moles of copper are bound per mole of fusion
protein. Similar ratios were obtained if refolded apo-fùsion protein was reconstitued with
copper. The amount of copper bound was found to be the same for both GST-WCBD and
WCBD, indicating that GST does not bind any copper and that the domain is able to bind 6 -
7 copper atoms. These results are consistent with similar observations made during the
characterization of Coxl7, which was also expressed as a fusion to GST (Srinivasan et al.,
1998).
3.2 J.3 6 5 ~ i n e blotting anak'ysis
In order to determine if WCBD is able to bind zinc and to m e r investigate the
rnetal binding properties of WCBD, a 6Stinc blotting assay was ernployed. Preliminary
experiments indicated that both GST-WCBD and WCBD were able to bind zinc under the
experimental conditions used. To determine the effect of pH on zinc binding, the assay was
carried out over a range of pH values fkom 6.5 - 9.0; these results are summarized in figure
3.4. Both GST-WCBD and WCBD are able to bind zinc over the entire pH range evaluated.
Progressive loss of zinc binding activity is only seen at the upper end of the pH range and
may be related to the pK, of the ligatiag atoms. GST alone did not bind appreciable amounts
of zinc, while the ERDBD (positive control) seemed to be much more sensitive to variations
in pH. The effect of reducing agents (DTT) on the binding of zinc was also examined.
These results are summarized in figure 3.5. The 6Szinc binding assay was carried out at pH
6.5 and 8.0 in either the absence or presence of DTT or just equilibrating the blot with DTT
and then rernoving it before addition of 65zinc. In the absence of DTT, zinc binding activity
was not observed at either pH value. However, strong zinc binding was observed when DTT
was used in the equilibration step only or if it was present throughout the assay. M e n
present throughout the assay, DTT was able to decrease the amount of non-specific binding
dramatically, as observed at pH 8.0. This is most likely due to the ability of DTT to chelate
non-specific and weakly bound zinc atoms. The requirement for DTT pretreatment suggests
that cysteine residues are directly involved in metal Iigation and that a fiee sulfhydryl is
required for metal ligation. This supports the notion that the two cysteine residues in the
HMA domain, strictly consewed in each of the metal binding domains of ATP7B, as well as
many bacterial heavy metal transporters, are crucial for metal ligation (O'Halloran, 1993;
Silver er al., 1989).
F C G E F C G E
Figure 3.4 Eflect of p H on the Bhding of "zinc. Samples of GST- WCBD (F), WCBD (C), GST (G), and the estrogen receptor DNA binding domain (E) were subjected to "zinc blotting analysis as described in section 2.5.2. In each case, blotting was perforrned in metal binding buffer at the indicated pH in the presence of 1 mM DTT.
G E C F M
G E C F G E C F M G E C F
Figure 3.5 Effect of Reducing Agents on the Binding of 6S~inc . Samples of GST-WCBD (F), WCBD (C), GST (G), and the estrogen receptor DNA binding domain (E) were subjected to "zinc blotting analysis, as described in section 2.5.2, at two pH values (6.5 and 8.0). M, molecular weight markers. To test the effects of reducing agents, DTT was either omitted fkom the assay (-DTT), included only during the equilibration step (+DTT/Eq), or included throughout the assay (+DIT).
3.2.2.4 Comptition "-'zinc b folomng ana&&
The ability of WCBD to bind copper and other transition metals was investigated
using a competition "zinc blot. The results of this analysis are presented in figure 3.6. AI1
competition 6Szinc blots were camed out using purified WCBD and DTT was present only in
the equilibration step. Several other transition metals were able to compete successfully with
zinc for binding to the domain. In particular, Cd(II), Au@), and H g 0 were found to have
the highest affinities for the domain relative to zinc. Both Mn@) and Ni(II) were not abte to
compete with zinc for binding to the domain when present in concentrations fiom 0.01 to O. 1
mM. Blotting with 63~ i ( I I ) directly instead of 6 S ~ n ( ~ ~ ) did not give rise to a signal on the
autoradiograph indicating Iack of binding to the domain. Co@), C r o , and Fe@) were the
weakest cornpetitors of the transition metals tested. The binding of zinc also seemed to be
specific, since a large excess of both Mg(I1) and Ca(I1) did not compete at al1 for zinc
binding. Results with copper were very dramatic and unique compared to the other metals
studied. At low concentrations, copper was able to decrease zinc binding by about 30%.
However, as the concentration of copper was raised, the afinity of copper seemed to increase
rapidly. This pattern was reproducible and independent of whether copper was presented in
the + l or +2 oxidation state, suggesting that the domain has similar afinities for both Cu(I)
and Cu(I1). The behavior of copper as a cornpetitor for zinc binding was similar to that
observed for the cooperative binding of ligands to proteins, indicating a possible cooperative
binding mechanism for copper which was not observed for the other transition metals tested.
0.00 1 0.0 1 o. 1 Competitor Concentration (mM)
100 90 80 70
60 50 40 30 20 10 O
Figure 3.6 Cornpetition of 6 5 ~ i n c Binding to WCBD Samples of purified WCBD were subjected to corn etition 6Szinc blotting analysis as descnbed in section 2.5.2.
6P In each case the final zinc concentration was 30 p M (-IO pCi). A, competition of 65 zinc binding with various transition metals. Al1 cornpetitor metals were added as the chloride salt at the indicated concentration. Ni(II), Mn@), Mg(lf), and Ca(II) showed no aEnity for the domain relative to zinc. B, competition of zinc binding with C u 0 and Cu(II). C u 0 was presented as CuClz while Cu(1) was presented as tetrakis(acetonitrile)copper(I) hexafluorophosphate. This complex was prepared in an argon-purged soIution of 2% acetonitrile.
- - - - 0 +
- + FetIII) + Cdrr)
- -0- cd(II) - -c cr(rrr)
- 6 Hg(W + Au(1Ii)
- + Ca(IQ - -X- Mg(1T)
0-
0.00 1 0.0 1 o. 1 1
Competitor Concentration (mM)
3.3 Surnmary and Discussion
We have expressed the amino-terminal metal binding domain of ATP7B as a tiision
to GST and studied its metal binding properties. We have shown that the domain binds
approximateiy 6 moles of copper per mole of protein regardless of whether GST is present or
absent. Using MAC, we have been able to detemine that the domain is able to bind a
variety of different transition metals with the apparent order of affinity:
Cu(I)/Cu(II)>Zn(II)>Ni(TI)>Co(II) under both denaturing and native conditions. Speci fic
binding of metalloproteins with intemal metal binding sites to IMAC columns under
denaturing conditions has also been observed for troponin T, which contains four repeated
rnetal binding motifs (Jin and Smiliie, 1994). In this assay, we did not observe binding of the
dornain to columns charged with either Fe(II) or F e o . This could indicate that the domain
had no affinity for iron or that the metal binding sites were unable to ligate iron while it was
bound to the MAC matrix, possibly due to steric effects.
The domain bound very strongly to columns charged with copper, as expected, and
could only be released with the specific copper chelator, BCS. Upon elution fÎom the
colurnn, the presence of Cu(1) in the protein was indicated by the formation of the reddish-
orange Cu(I)(BCS)-- cornplex, a surprising result considering that the column was charged
with Cu(I1) and non-reducing conditions were used. This observation suggests that the
domain is able to reduce copper from the +2 to +l oxidation state upon biading. This could
occur through the oxidation of a pair of cysteines in one domain followed by the binding of
copper to a reduced pair of cysteines in another domain. This gives rise to the possibility that
the domain could bind both C u 0 and Cu@) in vivo pnor to transport. The oxidation state of
the transported copper is not yet known, however there is one report which suggests that it
rnay be in the Cu@) state (Bingharn et al., 1996).
An in depth study of the metal binding properties of the domain using 65zinc blotting
techniques was performed. The results from this analysis have confmed and expanded
upon those obtained in the M A C experiments with two exceptions. Although the fusion
protein bound to [MAC columns charged with Ni(II), Ni(I1) was not able to compete
successfully with zinc for binding to WCBD in competition ''zinc blotting experiments. This
situation could aise if the affinity for zinc was much greater than that for nickel, in which
case nickel would continuously be competed away from the protein by zinc. This was tested
by blotting with 63nickel directly instead of 6Szinc. Binding of 63nickel to WCBD was not
observed, indicating that either the domain is not able to bind nickel or that the affinity for
nickel is extremely low. The converse situation was observed for iron, where binding was
not observed to iron-charged M A C columns, but iron was able to act as a cornpetitor for
zinc binding in competition blotting expenments.
The reasons for these discrepancies are not clear and they may be due to the inherent
differences in the two methods used. In MAC, the metal is immobilized on a matrix and the
protein must bind to the metal while it is bound to the matrix; in the blotting experiments, the
metal must bind to protein which is immobilized on nitrocellulose. in IMAC, the protein is,
in essence, 'sharing' the metal with the malrix, with some ligands coming fkom the matrix and
others fiom the protein in a temary form. Bearing this in mind, the results fiom the blotting
assays may more accurately reflect the binding properties of the domain, since al1 ligands
originate fiom the protein itself. The binding of transition metals other than copper to the
WCBD has been observed by snother laboratory (Lutsenko et al., 1997), and are in
agreement with what has been found in our studies. However, we have used two
independent methods and a wider range of metals then was used in the study by Lutsenko et
al. (1997) to assess the meta1 specificity of the WCBD.
The unique characteristics observed for copper as a cornpetitor may have important
consequences for the function of the domain in vivo. Although the blotting experiments
cannot be used to establish the presence of cooperative copper binding conclusiveIy, they are
very suggestive. The binding of zinc to the domain is not surprising since the Gly - Met -
Thr - Cys - Xaa - Xaa - Cys motif is also present in the bacterial zinc transporting ATPase,
ZntA (Rensing et al., 1997). What is not clear is how the specificity for copper is conferred
on the ATPase. If cooperative copper binding is taking place, it may be the basis of the
specificity. A cooperative binding mode1 for copper would require structural changes to take
place upon successive metal binding, which would bring the ligating residues to the
appropriate geometry for copper binding through tertiary folding of the protein. We have
studied the structural changes which occur in the WCBD using circular dichroism
spectroscopy and these results are presented in the following chapter.
CHAPTER 4
Structural Characterization of the Wilson Disease Copper Binding Domain And Its Meta1 Binding Sites
4.1 Introduction
The HMA domain is found in proteins fiom a wide range of organisms which are
invotved in the transport of a variety of heavy metals, including zinc, copper, cadmium and
mercury. In most lower organisms, one or two copies of the HMA are present; in bigher
organisms five or six copies are comrnon. Although it is common!y known that these
domains are involved in ligating the metal atorn prior to transport, little is known about the
structure of these domains and how the same domain can serve to ligate diverse metal atoms.
Furthemore, information regarding inter-domain interactions in multiple HMA domain
proteins (Menkes and Wilson disease ATPases) is practically nonexistent. The structural
information that is available is derived fiom studies on proteins with oniy one copy of the
HMA or fiom a single HMA excised fiom a protein containing multiple copies (Gitschier et
ai., 1998; Pufahl et al., 1997).
The NMR solution structure of the fourth HMA domain fiom the Menkes disease
protein complexed with silver(1) revealed Iinear coordination of the metal by the conserved
cysteine residues (Gitschier et al., 1998). Silver is known to prefer linear coordination and it
could be possible that the binding of silver to the domain may force linear coordination on
the metal binding site. Therefore, it is not clear if the conformation this domain adopts in the
presence of silver would be similar to that adopted in the presence of copper(I), which could
bind in either a linear, trigonal, or tetrahedral fashion. Additionally, the authors fouad that
the conformation of the apo-protein was essentially the same as the holoprotein. An XAS
study of the yeast copper chaperone ATX1, which contains one copy of the HMA, revealed
that copper iigation could be either Iinear or trigonal with al1 sulhr ligands (Pufahl et al.,
1997). In this system, it was proposed by the authors that both conserved cysteines and a
methionine were involved in Iigating the copper atom. Recently, a report has appeared fiom
an EXAFS study of the full amino-terminal metal-binding domain of the Menkes protein
fûsed to maltose binding protein (MBP). The authors of this report propose a cornpletely
linear ligation mode1 invotving only the conserved cysteine residues and also suggest a
copper - copper interaction (Ralle et al., 1998). However, this report does not give any
details regarding the copper:protein ratio or the purity of the sample, which would have
allowed usefùl conclusions to be draw from the data.
A recent study of the second metal binding domain fiom the Menkes disease protein
has shown that one domain does not undergo structural changes upon metal binding
(Harrison et al., 1999). The study of a single metal binding domain fiom either the Menkes
or Wilson disease protein can help in understanding the sub-structure of the intact domain;
however, these studies cannot reveal any information on how the entire domain rnay function
in vivo. It has been assumed that the conserved cysteine residues in the HMA repeats are the
only residues involved in Iigating the metal atom. However, analysis of the prirnary
sequence of the Wilson disease protein reveals six additional cysteine residues, evenly
distributed among the six HMA domains, which may play a role in metal ligation.
Furthemore, other potential Iigating residues such as histidine are also present in abundance
in the amino-termina1 metal-binding domain. Single domain studies or studies involving
single HMA proteins would not be able to detemine the potential fùnction of these or other
residues with regards to metal ligation. In order to address some of these questions, we have
begun a detailed structural study of the copper binding sites in the WCBD, using XAS (in
coIlaboration with Dr. Lawrence Que), in addition to analyzing the structural changes which
take place upon metal binding to the WCBD, using circular dichroism spectroscopy.
4.2 Results
4.2.1 X-ray absorption spectroscopy (X4S) anaiysis of GST- WCBD
A detailed analysis of the copper binding sites was carrîed out using XAS techniques
in collaboration with Dr. Lawrence Que, Jr. and Hua-Fen Hsu at the University of Minnesota.
Since the WCBD contains six copper binding sites, XAS techniques carried out on the fully
reconstituted domain (6 bound copper atoms) will give structural information which is
averaged over the six sites. To address the question of whether each copper binding site is
the same structurally, the XAS analysis was carried out on the fùsion protein containing
either 2, 3 or 5 copper atoms. The copper x-ray absorption near edge structure (XANES)
spectra for al1 three samples are shown in figure 4.1. Al1 three spectra are very similar and
exhibit a feature at 8983 eV which is consistent with the 1s -. 4p transition of Cu(Q ions
(Kau et al., 1987). The intensity of the transition at 8983 eV is correlated with the geometry
around the copper atom. Tetrathedrally coordinated copper(1) atoms have low intensity
transitionso while linear, two coordinate sites, have we!I resolved, high intensity transitions.
The observed transition for the WCBD is weaker than that of diagonal compounds but
stronger than that for trigonal compounds.
The extended x-ray absorption fine structure (EXAFS) spectra of the three samples
are shown in figure 4.2. The EXAFS spectra for al1 the samples are very similar both in k-
space and r'-space (fig. 4.2 A and B), which is consistent with the results of the XANES
spectra of the same samples. The ru-space spectra of al1 samples exhibit a prominent feature
centered around r' = 1.8 A (r - r' + 0.4 A) fiom a shell of sulfur scatterers. The best fit for the
first coordination sphere was obîained assuming a single shell of two s u l h atoms at 2.17-
2.19 A. A single shell with one or three sulfur atoms or splitting the shell into one sulfùr and
8970 8975 8980 8985 8990 8995 9000
Energy (eV)
Figure 4.1 Normalized Copper XANES Spectra For GST-WCBD with Vurious Amounts of Copper. XANES spectra were taken fiom lyophilized samples of GST-WCBD containing either 2 (2 Cu), 3 (3 Cu), or 5 (5 Cu) atoms. The amount of copper was quantified using neutron activation analysis. Cu(SR)z : [N(C3 H~)~]CU(SCLOH&; Cu(SR)3: [(Cs Hs)4P] [c~(SC6H5)3]-
Figure 4.2 EiAFS Spectra For GST- WCBD with Various Amoun fs of Copper. 2 Cu (solid line), 3 Cu (dotted line), 5 Cu (dash-dotted line). A EXAFS in k-space, B EXAFS in r'-space (r - r' + 0.4 A), C r'-space EXAFS for 5 Cu sample overlaid with that fiom Cu(SR)z (dot) and CU(SR)~ (diamond).
one low Z atom subshell at 2.17 - 2.19 and 1.9 - 2.1 A respectively, resulted in poorer fits.
The r'-space spectra of al1 samples also exhibit a minor peak at r' = 2.3 A which is not present
in the spectra of the mode1 compounds [ C U ( S C ~ ~ H ~ ~ ) ~ ] - and [CU(SP~)~]~- . A good fit for this
feature could not be obtained using either sulfbr or copper atoms.
4.2.2 Circuiar dichroisna anaiysis of GST- WCBD and WCBD
The confonnational dynamics of WCBD upon binding of metal were studied using
circular dichroism spectroscopy. Spectra of samples with increasing amounts of either
copper or zinc were recorded in the secondary structure region (250 - 200 nm) and the
aromatic region (250 - 400 nm). The spectra of the fusion protein and the WCBD alone
reconstituted with either copper or zinc are presented in figures 4.3, 4.4, 4.5 and 4.6. The
results obtained for the fusion protein are very simiiar to those of the WCBD alone, with
some exceptions. Addition of copper to the apo-fusion protein induced an increasc in
secondary structure (fig- 4.3 A). The magnitude of the increase was the greatest on going
from the apo state to the 2: 1 state and fiom the 2: 1 to 4: 1 state. Going fiom 4: 1 to 6: 1 or 8: 1
did not result in any further significant secondary structure changes. Signi ficant changes
were also observed in the aromatic region (fig. 4.3 B). The changes in this region pardleled
those observed in the secondary structure region with the Iargest changes occurring between
apo and 2: 1 and fiom 2: 1 to 4: 1. A large difference is also observed on going fiom 4: 1 to
6: 1 , whic h is not observed in the secondary structure region. No signi ficant changes were
observed on going fi-om 6: 1 to 8: 1 .
The changes observed upon the addition of zinc to the apo-fusion protein are very
different from those obtained with copper (fig. 4.4). Although the addition of zinc to the
apo-protein does cause an initial increase in secondary structure, additional molar equivalents
seems to cause an altemating increase and decrease in secondary structure (fig. 4.4 A). This
pattern is not o b s e ~ e d for copper, suggesting that the binding of zinc induces a structure
which is significantly different fiom that with copper. The differences between copper and
zinc are also observed in the aromatic region (fig. 4.4 B) where the pattern of changes is
significantly different from what is observed with copper. Substantial changes fkom the apo-
protein spectra are only observed on going fiorn apo to 4: 1 and fiom 4: 1 to 8: 1. The spectra
of the apo-fusion protein and the 2: 1 and 6: 1 zinc reconstituted protein are virtually identical.
Tbese observations, like those in the secondary structure spectra, suggest that the binding of
zinc to the dornain leads to a very different structure fiom that obtained with copper. This
finding may have significant implications for the metal selectivity of the pump in vivo.
The titration of the WCBD alone with copper produced results which were similar to
those observed with the fusion protein with some exceptions (fig. 4.5). A general increase in
secondary structure was observed upon progressive addition of copper. The magnitude of the
change was greatest between the apo form and the 2:1-form and to a lesser extent fiom the
4: 1-fom to the 6: 1-form (fig. 4.5 A). In contrast to the firsion protein spectra, addition of
copper equivalents above 6:l resulted in an apparent loss of secondary structure in the
domain. These differences could result fiom the presence and absence of GST, which may
be influencing certain structural changes in the fusion protein. The changes in the aromatic
region are also similar to those observed in the fusion protein, with some differences (fig. 4.5
B). The largest changes are observed on going fiom apo to 2: 1 and fkom 2: 1 to 4: 1 ; however,
in contrast to the fusion protein, only small changes were observed on
Wavelength (nm)
250 260 270 280 290 300 310 320 330 340 350 Wavelength (MI)
Figure 4.3 CD Spectra of GST- WCBD Titrared wirh Copper. The fusion protein was rcconstituted with copper to the indicated ratio and CD spectra recorded. A. Secondary structure region, B. Aromatic region.
Wavelength (MI)
250 260 270 280 290 300 310 320 330 340 350 Wavelength (nm)
Figure 4.4 CD Spectra of GST- WCBD Titrated with Zinc. The h i o n protein was reconstituted with zinc to the indicated ratio and CD spectra recorded. A. Secondary structure region, B. Aromatic region.
Wavelength (nm)
250 260 270 280 290 300 310 320 330 340 350 Wavelength (nm)
Figure 4.5 CD Spectra of WCBD Titrated wirh Copper. The W C B D was reconstituted with copper to the indicated ratio and CD spectra recorded. A. Secondary structure region, B. Aromatic region.
Wavelength (nm)
10 C
250 260 270 280 290 300 310 320 330 340 350
Wavelength (nrn)
Figure 4.6 CD Specfra of WCBD Titrafed with Zinc. The WCBD was reconstituted with zinc to the indicated ratio and CD spectra recorded. A. Secondary structure region, B. Aromatic region.
going tiom 4: 1 to 6: 1. There is also a significant change on going from 6: 1 to 8: 1, which was
not observed in the fbsion protein.
Titration of WCBD alone with zinc produced a pattem of conformational changes
(fig. 4.6) which were different fiom that of copper and resembled those obtained fiom the
addition of zinc to the apo &ion protein. The initial addition of zinc resulted in an apparent
loss of secondary structure. The addition of increasing amounts of zinc produced an
alternating increase and decrease in the amount of secondary structure present (fig. 4.6A).
This pattern was significantly different fiom that obtained with copper and resembled the
pattern found upon the addition of zinc to the fûsion protein. The aromatic region (fig. 4.6B)
also displayed features which were different fiom those obtained with copper. With the
copper titrations, an initial increase at 260 nm was observed fiom the apo to 2: 1 reconstituted
protein. At higher ratios this increase subsided and was replaced with an increase in the 290
nm range. For the zinc samples, there was only a small increase at 260 nm for the addition of
2 equivalents of zinc and at higher ratios no increases in the 290 nm range were obsewed.
These results suggest that the binding of zinc by the WCBD results in a conformation which
is significantly different fiom that with copper.
4.2.3 Estimation of Secondary Structure Content
The secondary structure content of the WCBD with various copper ratios (0,2,4,6, 8
metal atoms / mol protein) and of the fusion protein with zinc and copper (0, 2, 4,6, 8 metal
atoms / mol protein) were estimated as described in section 2.7.2. The results of this analysis
are presented in table 4.1. Each of the different metal ratios represents a separate protein
sample prepared fiom the same apo stock. Meta! concentrations were confirmed by N U ,
and protein concentrations were confirmed using the BCA protein assay.
4.2.4 Oxidutive refease of copper from GST- WCBD
The ability of oxidizing agents to liberate bound copper fiom the fùsion protein was
evaluated and these results are presented in figure 4.7. The results indicated that only
hydrogen peroxide was able to effect the release of approximately 2 copper atoms fiom the
domain after a 2 hour incubation. Addition of the oxidizing agent, ceric ammonium nitrate,
caused the protein to precipitate with no apparent loss of copper.
Table 4.1 Secondary Structure Estimation for Metal Titrated WCBD
and GST-WCBD Using SELCON and K2D
Metal : Protein Ratio Apo 2: 1 4: 1 6: 1 8: 1
CU-WCBD a-Helix (%) p-~heet' (%) p-Tms (%)
Other (%)
Zn-WCBD a-Helix (%) 6-~heet' (%) p-Tunis (%)
Other (%)
CU-GST-WCBD a-Helix (%) p-sheett (%) f3-Turns (%)
Other (%)
Other (%) 25 3 1 25 43 27 'values in brackets indicate f3-Sheet content estimated by the K2D program, al1 others are fiorn SELCON.
Control Hydrogen Ceric Ammonium Peroxide Nitratet
Figure 4.7 Oxidative Release of Bound Copper From WCBD. Copper reconstitued fusion protein was incubated for 2 hours in the presence of the indicated oxidizing agent. Remaining bound copper was quantified by NAA. tThis compound caused the protein to precipitate.
4.3 Summary and Discussion
The XAS studies of the WCBD with sub-stoichiometric amounts of copper have
provided a wealth of detailed structural information. The XANES spectra (fig. 4.1) M e r
confim the previous observation that copper bound to the WCBD is in the +1 oxidation state
by the presence of a feature at 8983 eV. This feature is attributed to a 1s -, 4p transition
which is characteristic of Cu(1) atorns. The intensity of this transition in the spectra of
WCBD is weaker than that for tinear copper thiolate complexes. This observation suggests a
distorted, two coordinate geometry around the copper atom. This distortion may arise fiom
protein conformational effects or the presence of a weak interaction from a third ligand.
There is some evidence of the latter due to the presence of the minor peak at r' = 2.3 A in the
EXAFS spectra. Good fits for this feature could not be obtained with either copper or su1 fur
atoms and, therefore, the identity of this scatterer remains undetermined.
This featwe is not present in the spectra of the mode1 compounds but is observed in
the EXAFS spectra of the Menkes protein (Ralle et al., 1998). The authors of this paper
suggest the possibility that this feature arises from the formation of copper clusters in the
domain. Although this is a definite possibility, the study by Ralle et al. is not detailed
enough to draw this conclusion. The XAS studies presented in this thesis are more detailed
since various ratios of copper were used. The results show that the feature at r' = 2.3 A
increases in intensity as greater amounts of copper are added. This could lend support to the
idea that copper clusters are forrned as more copper becomes bound to the domain; however,
the low intensity of this peak indicates that this is not a major form of the protein in these
samples. in preliminary experimcnts using samples which were reconstituted with copper in
the presence of TCEP and ascorbic acid, this feature is of greater intensity. The intensity of
the peak also rises as increasing amounts of copper are bound to the domain. Interestingly,
this peak is also a major feature in the spectra of non-lyophilized samples prepared in the
presence of DTT. It is possible that the domain can adopt multiple conformations under
different conditions. Further studies will be needed to establish the identity of the scatterer
which gives rise to this feature conclusively.
Fitting of the EXAFS data indicate that the first coordination sphere consists of two
sulfùr atoms with a Cu-S distance of 2.17 - 2.19 A. This distance is much shorter than those
in trigonal Cu(I)-thiolate complexes and is slightly longer than those of linear Cu(i)-thiolate
complexes (Coucouvanis et al., 1980; Fujisawa et al., 1998; Koch et al., 1984). Trigonal
Cu(1)-thiolate complexes have Cu-S bond distances of 2.27 - 2.38 A, whereas linear Cu(&
thiolate complexes have Cu-S bond distances of 2.14 - 2-15 A in structurally characterized
mode1 complexes. The Cu-S bond distances in the WCBD are similar to those found in the
Menkes pro tein (2.1 7 A) but are muc h shorter than those found in the yeast copper c haperone
ATX1 (2.25 A), which has only one HMA domain (Pufahl et al., 1997). This implies that the
copper sites in the WCBD are similar to those in the Menkes disease protein but significantly
different than that in ATXl. These observations indicate that the presence of multiple HMA
domains have an influence on the copper coordination environment- In light of these
findings, studies based on a single domain fkom the WCBD or those without carefully
characterized metal stoichiometries cannot provide an accurate representation of the domain
as a whole.
The structure dynamics of the WCBD (and GST-WCBD) upon titration with either
copper or zinc were studied using circula dichroism spectroscopy. Both the secondary
structure region (200 - 270 nrn) and the aromatic region (250 - 350 nm) were analyzed for
structural changes upon metal binding. The conformational changes which were obsewed
upon metal binding were similar for both the WCBD and GST-WCBD proteins (for the same
metal) with only minor differences. In the secondary structure region of proteins titrated
with copper (figs. 4.3, 4.5, panel A), the general observation is that the ellipticity increases
upon binding of greater amounts of copper. This increase suggests an increase or
stabilization of secondary structures relative to the metal free (apo) protein. This was
observed in both the fiision protein and the WCBD alone. In both cases, the magnitude of
the change is greatest between the apo fonn and the 2:l copper-fonn. However, in the
WCBD, another significant increase takes place on going fiom 4 to 6 copper atoms and this
is not observed in the fiision protein. Additionally, adding more than the stoichiometric
amount of copper to the WCBD resulted in an apparent destabilization of secondary structure
relative to the apo-protein. This phenomenon was not observed in the fùsion protein. These
differences are most likely the result of the presence of GST, which is able to form dimers in
solution. We have observed that, d- ing purification, the fusion protein is substantially more
soluble and Iess proue to aggregation than the WCBD alone. Furthermore, addition of excess
copper to concentrated solutions of the apo-WCBD usually results in the formation of a
precipitate, a phenomenon which is not obsewed with the fusion protein. Although
precipitate formation was not obsewed in the CD samples, soluble aggregates of the WCBD
may have been forrned at the higher copper ratios, leading to the apparent loss of secondary
structure observed in the CD spectra of the WCBD with an 8: 1 copper:protein ratio.
The changes in the aromatic region were also very similar between the fusion protein
and the WCBD. The changes which occwred in the aromatic region spectra upon addition of
copper paralleled those in the secondary structure region. The largest changes in spectral line
shape were observed on going fiom the apo-protein to the 2:l copper form and again fiom
the 2: 1 form to the 4: 1 copper fom. It is interesting to note that, in the WCBD, the 2: 1 and
4:1 copper forms seemed very similar in secondary structure but their tertiary structure
spectra were significantly different. A similar phenomena was also observed in the fiision
protein. This would seem to indicate that, although the secondary structure remains
relatively constant, the relative orientation of secondary structure elements within the domain
are changing, depending on how much metal is bound. These tertiary structure changes
could be the ba is of cooperativity in the domain where binding of copper to one site leads to
a modification of binding properties at another site. Cooperativity of copper binding to the
WCBD was suggested by competition zinc blotting experirnents using copper.
The spectra in the aromatic region *se fiom the rc-R' electronic transitions kom the
aromatic side chains of tyrosine, tryptophan, and phenylalanine residues. However, because
these residues contain complete or partial planes of symmetry, the transition a ise entirely
from interaction with neighboring groups (Wingfield and Pain, 1996). A detailed
interpretation of the spectra in this region is extremely difficult, since many of the transitions
overlap (table 4.2). In proteins which contain several of each kind of aromatic amino acid
(each in a different environment), deconvolution of the spectra to isolate contributions fiom
each residue is nearly impossible in the absence of either a three dimensional structure or
detailed mutational studies. Despite these difficulties, we can observe several patterns in
both the fiision protein and the WCBD, which occur upon copper binding. In going from the
apo to 2: 1 copper form a decrease (more positive) in ellipticity occurs in the 250 to 270 o . .
region. When copper is added to a 4: 1 ratio, the ellipticity in this region increases while the
ellipticity in the 270 - 290 nrn region decreases. The former region contains mainly
Table 4.2 CD Transitions of Aromatic Side chahst
--
Amino ~ x d Electronic Peak Transitions Wavelengths (nm)
Pheny lalanine 'Lb 262,268
Tyrosine
Tryptophan
far W 277,283
268,289 (shoulders)
' ~ a t a from (Strickland, 1974). ' ~ a ~ be seen as a low intensity peak or shoulder fiom a Trp in a nonpolar environment. n nu su al peak observed in lysozyme.
phenylalanine and tyrosine contributions, while the latter region contains mainly tryptophan
contributions (WingfieId and Pain, 1996). It is clear that the largest changes in the spectra
(both line shape and intensity) occur between the 2 and 4 copper fonns of the protein. This
indicates that a significant tertiary structure change is taking place afier the first few coppers
are bound, possibly reflecting the cooperative binding of copper aAer the fust few are bound.
This correlates well with the cornpetition zinc blotting studies (see section 3.2.2.4). At lower
copper concentrations zinc binding was reduced in a linear fashion and this was followed by
a sharp transition at hi@ copper concentrations, which is indicative of cooperative binding.
The disulfide bond can also make significant contributions to the ellipticity in CD
spectra because it does not contain an inherent plane of symmetry. The disulfide bond bas a
broad band of ellipticity in the 240 - 350 nm range and can augment or diminish the signal
from tyrosine and tryptophan residues (Wingfield and Pain, 1996). This M e r complicates
the interpretation of the spectra in this region. In cases where the disulfide makes a
significant contribution to the spectra, its presence is confirmed by ellipticity above 320 m.
This feature is present in both the fusion protein and the WCBD when they were titrated with
copper. It is most prominent in the apo form, since without copper and reducing agents the
18 cysteine residues in the domain will readily oxidize to form disulfide bonds. Furthemore,
this feature decreases as the amount of copper increases, since the cysteine residues are now
participating in metal ligation and are unavailable for disulfide formation. When six copper
atoms are bound, the ellipticity above 320 nm is nonexistent, suggesting that no disulfides are
present. The domain contains twelve conserved cysteine residues which are located in six
HMA domains (2 per domain) and are involved in ligating the metal. In addition, another six
cysteine residues are evenly interspersed between the HMA domains. It is not known
whether these form disulfides or are fiee in the native domain. The CD results indicate the
absence of disulfides in the fully copper-reconstituted state, indicating that either these
cysteines remain reduced or that they too are involved in metal ligation. However, at this
point we do not have any evidence to support one view over the other.
In contrast to the spectra obtained with copper, those obtained fiom the titration with
zinc are very different in both the secondaxy structure and aromatic regions (figs. 4.4 and
4.6). The addition of zinc to both the apo hision protein and the apo WCBD resulted in a
strange pattern of atternating increase and decrease of ellipticity which is in stark contrast to
the progressive increase in ellipticity observed for the copper samples. The same was mie
for the aromatic region, where the pattern of changes observed were not as significant and
did not resemble those for the proteins titrated with copper. In particular, the ellipticity
above 320 nm, which is indicative of the presence of disulfide bonds, did not decrease in a
Iinear fashion, as was observed with copper. Although we saw a decrease on going fkom the
apo to the 2 and 4 copper forms, no additional increase was observed on going fiom 4 to 6 or
8 coppers. These observations suggest that although zinc is able to bind to the domain (as
seen in IMAC and zinc blotting) it is not able to induce the same structure in the domain as
copper; this may be an important factor for the function of this dornain in vivo. Differences
in the conformation of the domain with zinc and copper may be due to the different
geometrical preferences of each metal. While zinc predominantly forms tetrahedral
complexes, copper can adopt conformations ranging fiom linear to distorted tetrahedral. This
distinction may play a role in conferring specificity of metal binding to the dornain.
Two computer programs SELCON and K2D were used to estimate the secondary
structure content of the fusion protein and the WCBD with various amounts of copper and
zinc (table 4.1). The SELCON program (Sreerama and Woody, 1994; Sreerama and Woody,
1993) uses CD data between 260 and 200 nm to predict a-helix, 6-structure, and fi-turns in
globular proteins. The program starts by arranging the proteins in the database in order of
increasing RMS difference fiom the CD spectrum to be analyzed and the spectra which are
most dissimilar are deleted. An initial guess of the unknown protein is made and this
conformation is included in the basis set, which is deconvoluted using singular value
decomposition (SVD). The secondary structure of the unknown protein is then caiculated.
The solution replaces the initial guess and the process is repeated until self-consistency is
attained. The K2D is a neural net program (Andrade et al., 1993; Merelo et al., 1994) which
uses a technique called proteinotopic mapping to calculate a-helix and 6-structure using CD
data between 240 and 200 am. It has been found to give the best estimate of f3-sheet content
using this data range and was used in conjunction with the SELCON program (Andrade et
al., 1993; Merelo et al., 1994).
The general trend in the WCBD titrated with copper was an increase in a-helix
content (-6%), a decrease in 6-sheet content (-IO%), no change in the fi-turn content and a
slight decrease in 'other' conformations (-2%). In contrast, titrating the WCBD with zinc
produced a fluctuation in the secondary structure content which followed the patterns seen in
the spectra. Both the trend and magnitude of changes were substantially different from those
produced with copper, further supporting the idea that these metals induce very different
conformations in the domain upon binding. The trends for the fusion proteins with either
copper or zinc were similar to those for the GST fiee domains. The absolute amount of each
secondary structure element was higher than for the WCBD alone due to the contributions of
GST to the spectra. The effect of copper binding seems to be one of stabilizing or increasing
a-helical content at the expense of f3-sbeet content. The binding of copper could rigidiS the
secondary structure around the metal binding site, stabilizing secondary structural elements
which, in the absence of metal, adopt a different conformation. Alternatively, stabilization
could be achieved by interaction of distant parts of the domain which are brought into close
proxirnity by the binding of copper.
These studies have shown that copper is bound fairly strongly to the domain;
however, at some point, the copper must be removed fiom the domain. We have investigated
the possibility that copper may be removed by oxidation. After incubation of copper WCBD
with either hydrogen peroxide or ceric ammonium nitrate, only 2 copper atoms were
liberated fiom the domain with hydrogen peroxide. Ceric ammonium nitrate resulted in the
precipitation of the protein; however, when copper was measured in the dialyzed precipitate,
al1 six copper atoms were still present. These results indicate that, at least with hydrogen
peroxide, it is possible to release some of the bound copper. The other copper atoms rnay be
located in regions inaccessible to the oxidizing agent (buried in the core) or involved in
structures which are resistant to oxidation. These results suggest the possibility that copper
could be transported in the +2 oxidation state. There is at least one study where ATP-
dependent transport of copper(1I) has been detected in the golgi apparatus of rat hepatocytes
(Bingharn et al., 1996). Further research into the form of the transported copper, as well as
the kinetics of copper binding to the amino-teminal domain, is needed.
4.3.1 Hypofhetical Model for the Function ofthe WCBD in vivo
The current work has provided insights into the mechanism by which metal is bound
to the WCBD and the structural changes which take place upon copper binding. Functional
studies, both in cultured cells and yeast mode1 systems have also provided important insights
into the role of the amino-terminal metal-binding repeats in the context of the whole ATPase.
Immunohistochemical studies in HepG2 cells have s h o w that, under normal, steady-state
conditions, the WND protein is Iocalized in the membrane of the tram-golgi (Hung et al.,
1997). However, if the concentration of copper was elevated in the media, a protein
translocation event was observed which resulted in the appearance of the WND protein in the
plasma membrane as well as an unidentified cytoplasrnic vacuolar location (Hung et al.,
1997). Furthemore, this translocation event was reversible and dependent on copper, since
removal of copper fiom the media resulted in the WND protein recycling back to the trans-
golgi. This phenomena has also been observed for the MNK protein and it is likely that these
proteins continuously recycle between the trans-golgi and the plasma membrane under steady
state conditions; elevated copper concentrations may shift the equilibrium toward plasma
membrane localization (Nagano et al., 1998; Petris et al., 1996).
It has also been shown that not al1 of the metal binding repeats are needed to mediate
the transport fûnction of the purnp (Forbes et al., 1999). Using a yeast complementation
assay, Forbes et al. (1999) were able to demonstrate that the mutation of metal repeats 1
through 4 did not affect the transport activity of the pump. Mutation of repeats 1 through 5
resulted in an approximate 5% drop in transport activity, while mutation of al1 six domains
totally abolished transport activity. This is in contrat to what has been found for the MNK
protein. In MNK, sequential mutation of the metal binding repeats does not result in a
significant loss of transport activity until the third motif is mutated (Payne and Gitlin, 1998).
This suggests that the metal binding repeats in the MNK protein are not functionally
equivalent and that motif three may play a central role in the binding and/or transport of the
metal. In the WND protein the data suggest that the metal binding repeats closer to the
membrane are more important than those at the amino-terminus- The metal binding sites in
the WND protein are also not functionally equivalent, since swapping the amino-terminal
three domains with the three domains closer to the membrane does not result in a fùnctional
protein (Forbes et al., 1999).
Taken together, these results can be used to construct a hypothetical model of how the
arnino-terminal metal-binding domain hnctions in the context of the whole ATPase (!Tg.
4.8). In this model, the WND protein would reside in the tram-golgi membrane under
normal physiological conditions where it would finction to pump copper fiom the cytoplasm
into the tram-golgi lumen. This copper could then be incorporated into secreted copper-
containing enzymes such as lysyl oxidase and ceruloplasmin. When unoccupied with copper,
the amino-terminal metal-binding domain interacts with the other cytosolic loops of the
ATPase (possibly the SEHPL motif region), preventing the transport of copper through the
pump. The binding of copper (most likely to the rnetal repeats closer to the trammembrane
segments) triggers a conformational change in the metal binding domain which disrupts or
changes its interaction with the cytosolic loops of the ATPase. This conformational change
could also promote the phosphorylation of the enzyme, initiating the transport cycle and
allow copper to be pumped into the tram-golgi lumen.
When the cytoplasmic concentrations of copper rise (as in copper overload), the
additional metal binding repeats would become progressively occupied with copper leading
to further conformational changes in the domain. These conforrnational changes would
somehow trigger the translocation of the WND (and MNK) proteins fiom the trans-golgi to
both the plasma membrane and a cytoplasmic vacuolar cornpartment. At the plasma
TGN Lumen
CopeOr ) Binding
Cytoplasm
1 A Extracellular 1
1. *[Cul* 2. Translocation
Figure 4.8 Hypothetical Model for the Function of WND in vivo. Under normal physiological conditions, the WND protein is localized in the bans-golgi network (TGN). When not occupied with copper, the metal binding domain interacts with the cytosolic loops of the ATPase. Binding of copper to the metal repeats closest to the transmembrane segments results in a conformational change wbich stimulates the phosphorylation of the ATPase and initiation of copper transport into the TGN lumen. Under conditions of elevated cytoplasmic copper, al1 of the metal binding repeats become occupied with copper resulthg in further conformational changes and the translocation of WND fiom the TGN to the plasma membrane (PM). At the PM, the WND protein would pump copper nom the cytoplasm into the extracellular space, lowering the cytoplasmic concentration of copper. As intracellular copper levels decrease, the metal binding repeats become progressively unoccupied and WND is recycled back to the TGN. A similar mechanism could also take place with the MNK protein.
membrane the WND and MNK proteins would be involved in pumping copper out of the ce11
in an effort to lower the cytoplasmic concentrations of copper. As the intracellular levels of
copper decrease, the metal binding domain would become progressively unoccupied leading
to the recycling of WND back to the tram-golgi membrane. The nature of the cytoplasmic
vacuolar compartment has not been analyzed. A recent study has shown that these
cytoplasmic vesicles are localized towards the hepatocyte canalicular membrane and may
provide a route for the excretion of copper into the bile (Scbaefer ez al., 1999). It is possible
that this compartment could be a lysosome, since copper pumped into lysosomes could then
be deposited into the bile through fùsion with the canalicular membrane. If true, this would
explain why biliary copper excretion in Wilson disease patients is severely compromised.
This point will have to be clarified by fùture studies.
Several details of the mechanism presented in figure 4.8 still remain to be elucidated.
For example, it is not known whether the copper atoms bound to the amino-terminal domain
are actually transported through the ATPase or if copper atorns fkom the cytoplasm are
transported. In either case, it seems clear from several studies of the WND and -MNK
proteins that not al1 the metal binding repeats are needed to allow transport activity (Hung et
al., 1997; Nagano et al., 1998; Petris et al., 1996). If copper bound to the amino-terminal
domain is transported, these observations suggest that copper is onIy transported from a few
of the metal binding sites (those closer to the membrane in WND, and those closer to the
amino-terminus in MNK). In this case, the other repeats could be Eûnctioning in a regulatory
role, acting as 'sensors' of intracellular copper concentration. If the amino-terminal bound
copper atoms are not transported, then the entire domain could be playing a role in regulating
the activity of the protein. The oxidation state of the transported copper atom is also not
known. Although we have shown that copper bound to the amino-terminal domain is in the
+ 1 oxidation state (DiDonato et al., 1997), some evidence suggests that copper is transported
in the +2 oxidation state (Bingham et al., 1996). If transported in the +2 oxidation state, the
amino-terminal bound copper would have to be oxidized prior to transport through the
membrane. This may be mediated by the protein itself or by an extemal oxidizing agent such
as hydrogen peroxide.
CHAPTER 5
General Discussion and Future Directions
5.1 General Discussion
The results presented in this thesis represent the fmt detailed characterization studies
of the copper binding domain fiom the Wilson disease copper transporting ATPase. These
studies have shown that the WCBD can bind a variety of transition metals in addition to
copper with varying affinities. This finding is intriguing since it leads to the question of
whether these other metals are traosported by the WND ATPase as well. This question has
not yet been addressed directly and will be an important topic for friture research. However,
we do know that the primary defect in Wilson disease is one of impaired copper emux Erom
the liver, leading to copper overload, which suggests that the main purpose of the WND
protein is to transport copper. Therefore, in the cytoplasm of the hepatocyte, what
mechanism does the protein employ to ensure specificity for copper? It is possible that the
protein never has an opportunity to interact with other metals in vivo due to their localization
in different cellular compartments. Ln recent years, a series of copper chaperones have been
identified, which are responsible for targetting copper to specific intracellular targets
(Valentine and Gralla, 1997). Similar proteins could exist for other transition metals to
facilitate their delivery to specific cellular compartments.
Altematively, the metal binding domain itself could be responsible for conferring
metal specificity on the protein. Bo& the 6Sz in~ blotting studies and the circula dichroism
studies have shown that the binding of copper to the WCBD may have some unique
properties which could result in specificity for copper as the transported ion. The
cornpetition 65zinc bloning studies have shown that a cooperative binding mode may exist for
copper, but not for the other metals. The structural changes in the domain induced by copper
binding are also very different fiom those induced by the binding of zinc. In the intact
protein, the binding of copper to the amino-terminal domain could trigger specific structural
changes which lead to the initiation of the transport cycle. Zinc would not be able to induce
the same conformation in the amino-terminal domain and, hence, the transport cycle could
not be initiated. XAS studies have shown that copper is ligated by two sulfur atoms in a
distorted linear arrangement. Zinc would not be able to bind to the domain in the same
manner since it prefers tetrahedral coordination and it is likely that the binding of zinc would
force the domain into a non-native conformation, an idea which is supported by our CD zinc
titration results.
The detailed structural data presented in this thesis, together with in vivo and in vitro
work of other groups, has allowed the formulation of a hypothetical mode1 for the function of
the LWD protein in vivo and the role the WCBD plays in this mechanism (fig. 4.8). In this
modei, the translocation of the protein, which is observed at elevated copper concentrations,
is rnediated by conformationai changes resulting from copper binding to the amino-terminal
domain. However, recent studies have indicated that this may not occur in the Menkes
protein. When the cysteine residues in HMA domains 1, 6, or 1-3 were mutated to serine in
the Menkes protein, the protcin was still able to relocate to the plasma membrane in response
to elevated copper (Strausak et al., 1999). However, if HMAs 4-6 or 1-6 were mutated, the
protein did not respond to elevated copper concentrations. Further analysis revealed that the
first four HMAs could be eliminated without loss of copper induced relocation; mutation of
the remaining two HMAs abolished its response to copper. Although the study by Strausak,
et al. (1999) evaluated the effects of these mutations on copper induced relocation, they did
not measure copper transport activity. An earlier report has shown that al1 transport activity
is lost wheo the cysteine residues in the fmt three HMA domains of the Menkes protein are
mutated (Payne and Gitlin, 1998).
These observations are in sharp contrast to similar studies performed on the Wilson
protein, which show that the first five HMA domains can be mutated without loss of copper
transport activity (Forbes et al., 1999). Taken together, these studies illustrate that the HMA
domains are not bctionally equivalent and that the amino-terminal domains of the Menkes
and Wilson proteins do not hnction in similar manners. In light of this, the results of the
study by Strausak, et al. (1999) cannot be extrapolated easily to the Wilson disease. Similar
studies on the Wilson disease protein will be needed to answer these questions.
5.2 Future Directions
The expression and characterization of the amino-teminal copper binding domain
from the WiIson disease copper transporting ATPase has given us invaluable information on
the possible fiuiction of this domain in the context of the entire protein. However, despite the
detailed analysis which has been carried out thusfar, several important questions concerning
the function of this domain in the transport of copper through the ATPase remain to be
addressed. Among these is the mechanism by which copper is delivered to the ATPase and
the subsequent steps which allow it to be transported across the membrane. The discovery of
a family of cytoplasmic copper chaperones has provided new avenues of research in this
area. Much of what is known about the fûnction of the copper chaperones has been derived
from complernentation studies in yeast. The yeast copper chaperone ATXI has been shown
to interact with the amino-terminal domain of ccc2, the yeast orthologue of WND, by yeast
two-hybnd analysis (Pufahl et al., 1997). The association of ATXl and ccc2 was also shown
to be dependent on the presence of copper. Recently, the human orthologue of ATXl has
been isolated and is known as ATOXl (formerly known as HAHI) (Klomp et al., 1997).
Due to the simiiarity between the ATXl and ATOXl proteins, an interaction bctween
ATOXI and the amino-terminal domain of the Wilson disease protein has been assumed to
exist, but has not been demonstrated experimentally .
In order to address the question of how copper is delivered to the domain in vivo, an
association behveen recombinant ATOX 1 and GST-WCBD wilt be demonstrated in vitro and
then characterized to determine the mechanism by which copper is transferred fiom ATOX 1
to the WCBD. The ba i s of the assay would consist of fornihg the complex in viîro and then
isolating it using glutathione affinity chromatography. Truncation and site-directed
mutagenesis studies will then De employed to isolate the region of the proteins which are
critical for cornplex formation and metal transfer. Expenments with the tnuicated protein
will establish if the chaperone is delivering copper to a specific HMA domain or has no
specificity for individual HMA domains. Together, these studies will help to define the
functional significance (if any) for each HMA repeat in the copper binding domain. The KD
values for copper binding to either the WCBD or ATOXl are also unknown. Solution ligand
binding experiments will be used to determine the dissociation constant for copper bound to
either the WCBD or ATOX1.
Another question, which is currently not addressed in the literature, is how copper is
transported across the membrane by the ATPase. Our studies have shown that once copper is
bound to the domain it is fairly resistant to oxidation and remains bound in the presence of
excess reducing agents, denaturants, and chelating agents such as EDTA. These observations
indicate that copper is bound tightly to the WCBD and lead to the question of how copper
can be removed from the WCBD once bound. We have investigated the possibility that
copper may be released by oxidation during the transport cycle. Prolonged exposure of the
the copper loaded WCBD to excess hydrogen peroxide resulted in the release of only two
copper atoms. In the intact protein, interactions between the amino-terminal domain and
other parts of the ATPase could also facilitate its release from the WCBD and its
translocation across the membrane. To M e r analyze this possibility, experiments must be
conducted using the entire ATPase. The Wilson disease ATPase couid be expressed in a
mammalian system and isolated in vesicles in a fùnctional form. Using this preparation,
detailed transport studies could be performed. If inverted vesicles could be prepared, such
that copper is pumped into the vesicte, the oxidation state of the transported copper could be
determined by examining the copper in the vesicles after the assay is complete. The
oxidation state of the copper inside the vesicles could easily be determined by electron
paramagnetic resonance (EPR)
The structural studies presented in this thesis have shown that significant structural
changes take place in the domain upon metal binding. Although both copper and zinc are
able to bind to the domain, the structures they induce upon binding are very different. This
difference could be the basis for the specificity of the ATPase. The HMA domain is found in
many proteins which bind a varie5 of metals (table 1.6), suggesting that it may be a general
metal binding motif. Studies on mtA, a zinc transporting ATPase in bacteria containing one
HMA domain, have shown that this protein c m transport cadmium as well as zinc, two
rnetals with similar ligation geometries (Rensing et al., 1997). Our cornpetition 65zinc
blotting studies have suggested that the binding of copper to the domain may have some
degree of cooperativity and that this property is unique to copper- In light of this
observation, we propose that the cooperativity observed for copper binding may contribute to
the specificity of the ATPase. To m e r examine the role of the metal binding domain and
the possible role it plays in conferring specificity to the ATPase, a chimeric protein could be
created containing the copper binding domain fiom the WND protein and the ATPase "core"
of zntA. The ability or inability of this chimera to transport copper could help confirm the
hypothesis that the WCBD is playing a part in determining the specificity of the transported
metal.
Although these experiments will give a very good understanding of how copper is
transferred and bound to the domain, a complete understanding of the fünction of this domain
wiIl require a detailed three-dimensional structure. For this reason, the three-dimensional
structure of the WCBD will be determined by x-ray crystallography. NMR will also be used
to determine the structure of a truncated fom (-35 kDa) of the domain, which contains only
KMA domains 4, 5, and 6, since biochemical snidies have indicated that these are of greater
importance to the function of the Wilson disease protein than the others (Forbes et al., 1999).
These structural studies, coupled with ongoing XAS experiments using copper and zinc
reconstituted proteins, will allow us to better define the role of this novel dornain in the
overall mechanism of the ATPase.
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