Biophysics 1

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Biophysics K. Banu Kse (50091012) 22.10.2010

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Human DNA topoisomerase I (70 kDa) in complex with the

indenoisoquinoline AI-III-52 and covalent complex with a 22 base

pair DNA duplex

1)

Structure

A) Primary

Chain A (Protein)

1 K K P K N K D K D K K V P E P D N K K K K P K K E E E Q K W K W W E E E R Y P E G I K W K F L E H K G P

V F A P P Y E P LYS LYS PRO LYS ASN LYS ASP LYS ASP LYS LYS VAL PRO GLU PRO ASP ASN LYS LYS LYS

LYS PRO LYS LYS GLU GLU GLU GLN LYS TRP LYS TRP TRP GLU GLU GLU ARG TYR PRO GLU GLY ILE LYS

TRP LYS PHE LEU GLU HIS LYS GLY PRO VAL PHE ALA PRO PRO TYR GLU PRO K K P K N K D K D K K V P

E P D N K K K K P K K E E E Q K W K W W E E E R Y P E G I K W K F L E H K G P V F A P P Y E P 174 61 L P

E N V K F Y Y D G K V M K L S P K A E E V A T F F A K M L D H E Y T T K E I F R K N F F K D W R K E M T NE E K N I LEU PRO GLU ASN VAL LYS PHE TYR TYR ASP GLY LYS VAL MET LYS LEU SER PRO LYS ALA GLU

GLU VAL ALA THR PHE PHE ALA LYS MET LEU ASP HIS GLU TYR THR THR LYS GLU ILE PHE ARG LYS ASN

PHE PHE LYS ASP TRP ARG LYS GLU MET THR ASN GLU GLU LYS ASN ILE L P E N V K F Y Y D G K V M K L

S P K A E E V A T F F A K M L D H E Y T T K E I F R K N F F K D W R K E M T N E E K N I 121 I T N L S K C D

F T Q M S Q Y F K A Q T E A R K Q M S K E E K L K I K E E N E K L L K E Y G F C I M D N H K E R I A N F ILE

THR ASN LEU SER LYS CYS ASP PHE THR GLN MET SER GLN TYR PHE LYS ALA GLN THR GLU ALA ARG

LYS GLN MET SER LYS GLU GLU LYS LEU LYS ILE LYS GLU GLU ASN GLU LYS LEU LEU LYS GLU TYR GLY

PHE CYS ILE MET ASP ASN HIS LYS GLU ARG ILE ALA ASN PHE I T N L S K C D F T Q M S Q Y F K A Q T E

A R K Q M S K E E K L K I K E E N E K L L K E Y G F C I M D N H K E R I A N F 181 K I E P P G L F R G R G N

H P K M G M L K R R I M P E D I I I N C S K D A K V P S P P P G H K W K E V R H D N K V T W L LYS ILE

GLU PRO PRO GLY LEU PHE ARG GLY ARG GLY ASN HIS PRO LYS MET GLY MET LEU LYS ARG ARG ILE

MET PRO GLU ASP ILE ILE ILE ASN CYS SER LYS ASP ALA LYS VAL PRO SER PRO PRO PRO GLY HIS LYS

TRP LYS GLU VAL ARG HIS ASP ASN LYS VAL THR TRP LEU K I E P P G L F R G R G N H P K M G M L K R R

I M P E D I I I N C S K D A K V P S P P P G H K W K E V R H D N K V T W L 241 V S W T E N I Q G S I K Y I M

L N P S S R I K G E K D W Q K Y E T A R R L K K C V D K I R N Q Y R E D W K S K E M K V R VAL SER TRP

THR GLU ASN ILE GLN GLY SER ILE LYS TYR ILE MET LEU ASN PRO SER SER ARG ILE LYS GLY GLU LYS

ASP TRP GLN LYS TYR GLU THR ALA ARG ARG LEU LYS LYS CYS VAL ASP LYS ILE ARG ASN GLN TYR ARG


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GLU ASP TRP LYS SER LYS GLU MET LYS VAL ARG V S W T E N I Q G S I K Y I M L N P S S R I K G E K D W

Q K Y E T A R R L K K C V D K I R N Q Y R E D W K S K E M K V R 301 Q R A V A L Y F I D K L A L R A G N E

K E E G E T A D T V G C C S L R V E H I N L H P E L D G Q E Y V V E F D F L G K D GLN ARG ALA VAL ALA

LEU TYR PHE ILE ASP LYS LEU ALA LEU ARG ALA GLY ASN GLU LYS GLU GLU GLY GLU THR ALA ASP THR

VAL GLY CYS CYS SER LEU ARG VAL GLU HIS ILE ASN LEU HIS PRO GLU LEU ASP GLY GLN GLU TYR VAL

VAL GLU PHE ASP PHE LEU GLY LYS ASP Q R A V A L Y F I D K L A L R A G N E K E E G E T A D T V G C C S

L R V E H I N L H P E L D G Q E Y V V E F D F L G K D 361 S I R Y Y N K V P V E K R V F K N L Q L F M E N K

Q P E D D L F D R L N T G I L N K H L Q D L M E G L T A K V F R T Y N SER ILE ARG TYR TYR ASN LYS VAL

PRO VAL GLU LYS ARG VAL PHE LYS ASN LEU GLN LEU PHE MET GLU ASN LYS GLN PRO GLU ASP ASP

LEU PHE ASP ARG LEU ASN THR GLY ILE LEU ASN LYS HIS LEU GLN ASP LEU MET GLU GLY LEU THR ALA

LYS VAL PHE ARG THR TYR ASN S I R Y Y N K V P V E K R V F K N L Q L F M E N K Q P E D D L F D R L N T

G I L N K H L Q D L M E G L T A K V F R T Y N 421 A S I T L Q Q Q L K E L T A P D E N I P A K I L S Y N R A N

R A V A I L C N H Q R A P P K T F E K S M M N L Q T K I D A ALA SER ILE THR LEU GLN GLN GLN LEU LYS

GLU LEU THR ALA PRO ASP GLU ASN ILE PRO ALA LYS ILE LEU SER TYR ASN ARG ALA ASN ARG ALA

VAL ALA ILE LEU CYS ASN HIS GLN ARG ALA PRO PRO LYS THR PHE GLU LYS SER MET MET ASN LEU

GLN THR LYS ILE ASP ALA A S I T L Q Q Q L K E L T A P D E N I P A K I L S Y N R A N R A V A I L C N H Q R

A P P K T F E K S M M N L Q T K I D A 481 K K E Q L A D A R R D L K S A K A D A K V M K D A K T K K V V ES K K K A V Q R L E E Q L M K L E V Q A T D R E E N K Q L YS LYS GLU GLN LEU ALA ASP ALA ARG ARG

ASP LEU LYS SER ALA LYS ALA ASP ALA LYS VAL MET LYS ASP ALA LYS THR LYS LYS VAL VAL GLU SER

LYS LYS LYS ALA VAL GLN ARG LEU GLU GLU GLN LEU MET LYS LEU GLU VAL GLN ALA THR ASP ARG

GLU GLU ASN LYS GLN K K E Q L A D A R R D L K S A K A D A K V M K D A K T K K V V E S K K K A V Q R L

E E Q L M K L E V Q A T D R E E N K Q 541 I A L G T S K L N Y L D P R I T V A W C K K W G V P I E K I Y N K

T Q R E K F A W A I D M A D E D Y E F ILE ALA LEU GLY THR SER LYS LEU ASN PTR LEU A SP PRO ARG ILE

THR VAL ALA TRP CYS LYS LYS TRP GLY VAL PRO ILE GLU LYS ILE TYR ASN LYS THR GLN ARG GLU LYS

PHE ALA TRP ALA ILE ASP MET ALA ASP GLU ASP TYR GLU PHE I A L G T S K L N Y L D P R I T V A W C K

K W G V P I E K I Y N K T Q R E K F A W A I D M A D E D Y E F

Chain B (DNA)

1 A A A A A G A C T T

Chain C (DNA)

1 C G A A A A A T T T T T

Chain D (DNA)

1 A A A A A T T T T T C G A A G T C T T T T T


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b) Secondary

Chain A (Protein)






E N V K F Y Y D G K V M K L S P K A E E V A T F F A K M L D H E Y T T K E I F R K N F F K D W R K E M T N

E E K N I LEU PRO GLU ASN VAL LYS PHE TYR TYR ASP GLY LYS VAL MET LYS LEU SER PRO LYS ALA GLU



S P K A E E V A T F F A K M L D H E Y T T K E I F R K N F F K D W R K E M T N E E K N I 121 I T N L S K C DF T Q M S Q Y F K A Q T E A R K Q M S K E E K L K I K E E N E K L L K E Y G F C I M D N H K E R I A N F ILE




















Q P E D D L F D R L N T G I L N K H L Q D L M E G L T A K V F R T Y N SER ILE ARG TYR TYR ASN LYS VALPRO VAL GLU LYS ARG VAL PHE LYS ASN LEU GLN LEU PHE MET GLU ASN LYS GLN PRO GLU ASP ASP

LEU PHE ASP ARG LEU ASN THR GLY ILE LEU ASN LYS HIS LEU GLN ASP LEU MET GLU GLY LEU THR ALA






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A P P K T F E K S M M N L Q T K I D A 481 K K E Q L A D A R R D L K S A K A D A K V M K D A K T K K V V E

S K K K A V Q R L E E Q L M K L E V Q A T D R E E N K Q LYS LYS GLU GLN LEU ALA ASP ALA ARG ARG





T Q R E K F A W A I D M A D E D Y E F

Chain B (DNA)


Chain C (DNA)


Chain D (DNA)


c) Tertiary

Chain A (Protein)






E N V K F Y Y D G K V M K L S P K A E E V A T F F A K M L D H E Y T T K E I F R K N F F K D W R K E M T N

E E K N I LEU PRO GLU ASN VAL LYS PHE TYR TYR ASP GLY LYS VAL MET LYS LEU SER PRO LYS ALA GLU



S P K A E E V A T F F A K M L D H E Y T T K E I F R K N F F K D W R K E M T N E E K N I 121 I T N L S K C D

F T Q M S Q Y F K A Q T E A R K Q M S K E E K L K I K E E N E K L L K E Y G F C I M D N H K E R I A N F ILE






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Q P E D D L F D R L N T G I L N K H L Q D L M E G L T A K V F R T Y N SER ILE ARG TYR TYR ASN LYS VAL

PRO VAL GLU LYS ARG VAL PHE LYS ASN LEU GLN LEU PHE MET GLU ASN LYS GLN PRO GLU ASP ASPLEU PHE ASP ARG LEU ASN THR GLY ILE LEU ASN LYS HIS LEU GLN ASP LEU MET GLU GLY LEU THR ALA







A P P K T F E K S M M N L Q T K I D A 481 K K E Q L A D A R R D L K S A K A D A K V M K D A K T K K V V E

S K K K A V Q R L E E Q L M K L E V Q A T D R E E N K Q LYS LYS GLU GLN LEU ALA ASP ALA ARG ARG





T Q R E K F A W A I D M A D E D Y E F ILE ALA LEU GLY THR SER LYS LEU ASN PTR LEU ASP PRO ARG ILE

THR VAL ALA TRP CYS LYS LYS TRP GLY VAL PRO ILE GLU LYS ILE TYR ASN LYS THR GLN ARG GLU LYS

PHE ALA TRP ALA ILE ASP MET ALA ASP GLU ASP TYR GLU PHE I A L G T S K L N Y L D P R I T V A W C K

K W G V P I E K I Y N K T Q R E K F A W A I D M A D E D Y E F 765

Chain B (DNA)


Chain C (DNA)



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Bi h si s K Banu K s

S

y

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Chain D (DNA)


Quate nary

Annotate PDB QuaternaryStructure Asse ly:1

Hetero Tetrametric

4 subunits of4 distinct poly

ers entities (5 molecules in total including ligands)


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d) Molecular Function of 1tl8

Synthesis and mechanism of action studies of a series of norindenoisoquinoine topoisomerase I

poisons reveaan inhibitor with a f

ipped orientation in the ternary DNA-enzyme-inhibitor comp

ex as

determined by X-ray crysta ographic ana

ysis.

91.8% (516/562) of all residues were in favored (98%) regions.


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98.4% (553/562) of all residues were in allowed (>99.8%) regions.

There were 9 outliers (phi, psi):

202 LYS (83.2, 62.2)

212 PRO (-56.6, 88.3)

213 GLU (4.7, 121.8)

236 GLU (-8.1, -51.9)

407 ASP (-51.7, 93.5)

467 SER (-1.5, 133.6)638 LYS (-37.2, 91.0)675 MET (86.1, 101.8)

724 LEU (65.5, 102.3)

DNA topoisomerase 1 is an enzyme that in humans is encoded by the TOP1 gene.

This gene encodes a DNA topoisomerase, an enzyme that contro s and a ters the topo ogic states of

DNA during transcription. This enzyme catayzes the transient breaking and rejoining of a sing

e

strand of DNA which a ows the strands to pass through one another, thus a

tering the topo

ogy of

DNA. This gene isoca

ized to chromosome 20 and has pseudogenes which reside on chromosomes 1

and 22.

We demonstrate that five topoisomerase I (Top 1) inhibitors (two indenoisoquinoines, two

camptothecins, and one indo ocarbazo e) each interca ate between the base pairs f anking the

ceavage site generated during the Top1 cata

ytic cyc

e and are further stabi

ized by a network of

hydrogen bonds with Top1. The interfacia inhibition paradigm described for Top1 inhibitors can be

genera ized to a variety of natura products that trap macromo ecu ar comp exes as they undergo

cata ytic conformationa changes that create hotspots for drug binding. Stabi ization ofsuch

conformationastates resu

ts in uncompetitive inhibition and exemp

ifies the re

evance ofscreening

for igands and drugs that stabi ize (trap) these macromo ecu ar comp exes

(DNA Topoisomerase I Inhibitors: Chemistry, Biology, and Interfacial In hibition/Yves Pommiera/LaboratoryofMolecular

Pharmacology, Centerfor Cancer Research, NationalCancer Institute, NationalInstitutes ofHealth, Bethesda, Maryland

Chem. Rev., 2009)

e)

Crystallization Experiments

Method VAPOR DIFFUSION, SITTING DROP pH 6.4 Temperature 289.0 Detais PEG 8000, MES,

Ammonium Sufate, pH 6.40, VAPOR DIFFUSION, SITTING DROP, temperature 289K


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Crystl

t

/ Unit Cell

Length

Angle ()

a = 56.95 = 90

b = 114.14 = 94.18

c = 73.5 = 90

2) DNA TOPOLOGY

The double-st ded st ucture of DNA has many implications for biological

function. Replication, at first appeared facilitated because genetic information isencoded t ice in the DNA structure, once on each strand, however, the two parental

strands must be separated and unwound to be copied.Transcriptional enzymes must decide which oftwo complementary strands

contains the correctinformation to copy. Transcription also involves transientunwinding ofthe DNA helixin a local region in orderto be able to copy one strand.

Metabolic events involving unwinding impose great stress on the DNA because

ofthe constraints inherentin the double helix. Today we will discuss the topology of

DNA and tomorrow we will cover enzymes that can alterthe topological state of DNA


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without changing its primary structure. These enzymes are called DNA

topoisomerases.In addition to the requirement to unwind DNA for replication and for

transcription, there is an absolute requirement for the correct topological tension in theDNA (super-helical density) in order for genes to be regulated and e pressed normally.

This supercoiling or writhing of circular DNAs was a result of the DNAs being

underwound with respect to the rela ed form of DNA. There are actually fewer turns in the

DNA heli than one would e pect given the natural pitch of DNA in solution(10.4 base pairs per turn).

When a linear DNA is free in solution it assumes a pitch which contains 10.4

base pairs per turn. This is less tightly wound than the 10.0 base pairs per turn in the

Watson and Crick B-form DNA.

In order to understand the origin of supercoiling; imagine a linear DNA 5200

base pairs in length. If the DNA were in the B-form one would e pect the two strands of

the heli to be wrapped around each other 500 times (5200 bp/10.4 bp/turn). Imagine alinear DNA in which the two ends become connected to form an open circle. This isreferred to as a rela ed circular DNA. On the other hand, if the linear DNA were

unwound 10%, say 50 turns, before its ends were joined, then the DNA molecule

would be under stress. When the molecule is free in solution it will coil about itself in

space as the two strands simultaneously twist about each other in order to return to

equilibrium value of 10.4 base pairs per turn.

DNA that is underwound is referred to as negatively supercoiled. The helices

wind about each other in a right handed path in space.

DNA that is overwound also will rela and assume a supercoiled conformation

but this is referred to as a positively supercoiled DNA heli . Positively coiled DNA hasits DNA helices wound around each other in a left-handed path in space.

The total number of times one strand of the DNA heli is linked with the otherin a covalently closed circular molecule is known as the linking number Lk.


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1. The linking number is only defined for covalently closed DNA and its value is

fi ed as long as the molecule remains covalently closed.

2. The linking number does not change whether the covalently closed circle is

forced to lie in a plane in a stressed conformation or whether it is allowed to

supercoil about itself freely in space.

3. The linking number Lkof a circular DNA can only be changed by breaking a

phosphodiester bond in one of the two strands, allowing the intact strand to pass

through the broken strand and then rejoining the broken strand.4. Lkis always an integer since two strands must always be wound about each

other an integral number of times upon closure.The linking number of a covalently closed circular DNA can be resolved into

two components called the twists Tw and the writhes Wr.Lk= Tw + Wr

The twists Tw are the number of times that the two strands are twisted about

each other while the writhes Wr is the number of times that the DNA heli is coiledabout itself in three-dimensional space.

Unlike the Twist and the Linking number, the writhe of DNA only depends on

the path the heli a is takes in space, not on the fact that the DNA his two strands. If

the path of the DNA is in a plane, the Wr is always zero. Also if the path of the DNAheli were on the surface of a sphere (like the seams of a tennis ball or base ball) then

the total Writhe can also be shown to be zero.Writhes


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DNA GEOMETRY

A-DNA / B-DNA

When DNA fibres are formed at low humidity DNA assumes a less hydrated form. (A-form)This structure was determined at the same time as the B-form. This too is a double heli

structure, but is formed at a lower hydration than B-DNA (i.e.


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Oligonucleotide Structures

More recently small, self-complimentary pieces of DNA have been co-crystallised and theirstuctures determined by X-ray crystallography. These are true crystalls and the positions of

each individual atom and base-pair can be determined. This contrasts with the fibre difraction

patterns which only give an average picture for all base pairs. Analysis of these crystalls has

shown that DNA can have quite large local deviations from the canonical Watson and Crick

structure and that these deviations appear to be dependent on the sequence of base pairs. The

crystal structure of the B-DNA decamer:

shows there are:

y About 10.1 base pairs/turn

y An average helical twist of 35.6o

y An average rise/residue of 0.34nm


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These average values are quite close to the values for those for B-DNA determined from fibre

difraction. However there are large variations from these average values for individual basepairs in the sequence, such that:

y The rise/residue varies from 0.30nm to 0.42nm

y The helical twistvaries from 32o

to 40o

y The sugar conformation varies, some are C3'-endo, some are C2'-endo, and some assume an intermediate conformation

y The base roll (angle to heli a is) varies

y Individual bases in a base pair have propeller twist

y Bases in a base pair are at a dihedral angle to each other

The variations in geometry appear to be sequence specific, purines favouring the C2'-endo

sugars, and pyrimidines the C3'-endo sugars. The conformation of a residue is also affected by

what it's neighbours conformation is (i.e. the AT pairs in the middle have a slightly different

conformation to those ne t to the GC's).

Use the mouse to rotate and zoom the image and convince yourself of the changes in propellertwist and dihedral angle. The changes in sugar conformation are not quite so easy to spot.

The heli is curved by appro imately 19o and not straight. (This is not easy to spot either if

you have not had a lot of e perience looking at structures of this type.) DNA helices are

reasonably fle ible to bending about small angles, so it is not known whether the curve seen is

due to natural conditions or an artifact of the crystallisation process.

There is a row of tightly bonded water molecules in the major groove in the AT-rich region. It

is not possible to say if these water molecules are present in native DNA as fibre difraction

does not enable water molecules to be identified. It is thought these water molecules may

provide a "spine of hydration" which stabilises the structure in the B-form and may be lost inthe transition to A-form. This is speculation however.

DNA is easily deformed, and does not have a precisely regular structure. Geometric variations

apear to be sequence specific...

If the geometry of DNA changes according to sequence, there may be a

geometrical mechanism for recognition of sequence.A protein could initially recognise a particular sequence from the shape

of the DNA it binds to.


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Z-DNA

The third and final geometry of DNA is that of the so-called Z-DNA.

This is sometimes seen when the sequence is of alternating purines and pyrimidines and wasfirst seen in the structure of the alternating deo ynucleotide dCpGpCpGpCpG.

If this structure displays the hydrogens-white dots- use the pull down menu to turn them off.(OPTIONS submenu)

The Z structure was first shown in oligonucleotides, and an anti-parallel double heli forms ...

BUT the heli is:

y Left handed

y 12 base pairs/turn

y Pitch is 4.5nm per turn

y Deep minor groove

y Little or no major groove

This structure is formed by rotating the N-glycosyl bonds of G residues 180o with respect to

their conformation in B-DNA. In Z DNA all of the purines are in the "syn" conformation and

all the pyrimidines are in the "anti" conformation. Because the purines are "flipped" because

of the 180o

rotation the pyrimidines must also "flip" to keep the base pairing intact. However

pyrimidine nucleotides do not readily assume the "syn" configuration because of steric

hinderance between the o y group at position 2 of the pyrimidine and the atoms of the sugar.


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In order to keep the base pairs intact the whole nucleoside (base plus sugar) has to flip 180o.

Despite these quite drastic conformational changes it is topologically possible for the G basesto go syn and the C nucleosides to rotate 180

owithout breaking and reforming the hydrogen

bonds. This means that the B to Z structural transition can occur without any separation of theheli .

It is not known whether Z-DNA is biologically significant, although antibodies raised tooligonucleotides of this structure, have bound to some DNA regions. At least one DNA

binding protein has been found which is specific to Z-DNA. This form of DNA is only found

in high salt concentrations, because a high concentration of cations are required to maintain

the stability between the phosphate backbones (which are in close pro imity to each other inZ DNA).

Source

http //www.pdb.org/pdb/exp ore/exp ore.do?structureId=1TL8

http //pubs.acs.org/doi/abs/10.1021/cr900097c

http//www.ebi.ac.uk/pdbsum/1t

8

http//books.goog

e.com.tr/books?id=WGBAGyzvQOUC&pg=PA25&dq=DNA+TOPOLOGY+AND+GEO

METRY&source=gbs_toc_r&cad=4#v=onepage&q=DNA%20TOPOLOGY%20AND%20GEOMETRY&f=fa

se

http //www.ima.umn.edu/2007-2008/T9.15.07/activities/O son-Wi ma/IMA_tutoria _1.pdf

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Biophysics 1