Structural features that govern enzymatic activity of Carbonic Structural features that govern enzymatic activity of Carbonic

anhydrase in a low temperature adapted fish anhydrase in a low temperature adapted fish

Chionodraco hamatusChionodraco hamatus

 Stefano Marino *, Kuniko Hayakawa* +, Keisuke Hatada+, Maurizio Benfatto+, Antonia RizzelloMichele Maffia, Luigi Bubacco*  * Department of Biology, University of Padova, Padova, Italy+ Laboratori Nazionali di Frascati dell’INFN - INFN, c.p. 13, Frascati, Italy Department of Biology, University of Lecce, Lecce, Italy

Roles of Zinc:Roles of Zinc:

- Electrostatic catalist- Electrostatic catalist: stabilize the negative charged transition from COCO22 to HCO to HCO33¯̄

-- Lower pKa Lower pKa of the coordinated water ( ( pH 7pH 7: coordinated : coordinated OHOH-- is an excellent nucleophile excellent nucleophile ) )

Carbonic Anhydrase (CA):Carbonic Anhydrase (CA):

Structure of the reaction centeStructure of the reaction center (RC):r (RC):

Primary ligands:

His 94, 96,119 His 94, 96,119 ;; HOH HOH

Thr199Thr199: H-bond with HOH

Glu106Glu106: H-bond with Thr199

Mutations in these RC positions Mutations in these RC positions >> >> loss loss // strong strong decrease of decrease of enzymatic enzymatic activity activity

Activity in f(T) (Maffia,2002):C.hamatus,T. bernacchii, A. anguilla

C.hamatus:C.hamatus:

1) loss of activity for1) loss of activity for T higher than 30°CT higher than 30°C

Chionodraco hamatus Chionodraco hamatus (Icefish) Carbonic Anhydrase:(Icefish) Carbonic Anhydrase:

C.hamatus:C.hamatus: Antartic fish lacking Haemoglobin and Red blood cells

Aims of present study on C. hamatus Aims of present study on C. hamatus CA (CAice)CA (CAice)::

1) structure of Reaction Centre (RC)1) structure of Reaction Centre (RC) in Icefish in Icefish, compared with Human carbonic anhydrase II (CA2h) as a reference structure

2) 3D structure2) 3D structure of CA icefish

Tools :Tools :

1)1) XAS spectroscopy at the k-edge of the RC : XANES spectraXANES spectra

2) Molecular Modelling 2) Molecular Modelling

Sequence analysis:Sequence analysis:

High activity cytosolic CA conservation in :High activity cytosolic CA conservation in :

Vertebrata: an average of 60 % aminoacidic identity

FishFish: an average of 72 %72 % of aacidic identity

Mammalian CAIIMammalian CAII:: an average of 74 %74 % of aacidic identity

NBNB: a) : a) MammaliaMammalia: : 3 cytosolic isoforms (CAI, CAII,CAIII); higher activity isoform is CAII b) Fish Fish high activity CA is more similar to CAII (67% vs 60% CAI, 57% CAIII)

expecially in RC (89%, vs 80% CAI, 70% CAIII)

>> so we consider mammalian CAII as our reference mammalian CA>> so we consider mammalian CAII as our reference mammalian CA

conservation

0

0,2

0,4

0,6

0,8

1

1,2

10 15 20 25 s_canale

Zn distance

% id

infraclass: teleostei

class: mammalian

subphylum : vertebrata

Conservation in Mammalia (CAII), Teleostei, VertebrataConservation in Mammalia (CAII), Teleostei, Vertebrata

'extended RC' (aa within 10 aa within 10 ÅÅ from Zinc) : % id % id ~ 90 ~ 90 % % for vertebratevertebrate CACA

'XANES RC' (~ ~ 7 Å from Zinc)Å from Zinc) : % id = 100% % id = 100% for vertebrate for vertebrate CACA

15 15 ÅÅ

ZN

10 10 ÅÅ

Template selection: PDB reference for computationsTemplate selection: PDB reference for computations

Metap serverMetap server: : 3D-jury3D-jury scoring/ranking alghoritm scoring/ranking alghoritm

•1flj CA III S- glutathiolated Rattus Norvegicus R[Å]=1.80Å

•1v9i CA II bos taurus, Q 253>C R[Å]=2.5 Å

•2cba CA II Homo sapiens (CA2h) R[Å]=1.54Å

•12ca CA2h Ala 121> Val 121 R[Å]=2.40 Å

•1hcb CAI Homo sapiens, with bicarbonate R[Å]=1.54Å

2cba (CAII) vs 1flj (CAIII):2cba (CAII) vs 1flj (CAIII):

> 2cba is best resolved (1.54 A)> 2cba is best resolved (1.54 A) > > CAiceCAice RCRC : more similar to mammalian : more similar to mammalian CAIICAII thanthan CAIIICAIII

>> Choice of 2cba>> Choice of 2cba

XANES experimental dataXANES experimental data

A = Icefish CA (CAice)

B = Human CA (CA2h)

THE MXAN METHOD :THE MXAN METHOD :

We generate hundred of theoretical spectra by moving atomic coordinates

(The potential is calculated at each step )

By comparison with exp. data we can fit relevant structural parameters

Minimization of error function

• Initial geometrical configurations (2cba)

• Exp. data (Xanes spectra)

Structural parameter optimization

Structural simulation: 64 atoms of the reaction center (~ 7 Å from Zinc )

(Zinc, 8HOH,Glu106,Thr199,Thr200, 3His leganti, Phe95, Val143, Glu117):

>> structural parameters with more impact

1) HOH263-Zn: distance and Theta angle

2) Thr199Thr199: Theta angle (Oδ) and distance

3) Coordinated His: distance

N

NN

N

Zn

N

N

OHθ

N

NN

N

Zn

N

N

OHθ

Final fitting for CA2h and CAice

Bestfit CA2hCA2h (Х2 = 4,04)

Bestfit CAiceCAice (Х2 = 4,44)

Final structural data Final structural data

1) HOH263: significantly closer to Zinc in CA2h

2) O2) Oγ (Thr199) :γ (Thr199) : closer in CA2h, closer in CA2h, consistentlyconsistently with the closer HOH263 with the closer HOH263

CAhumanCAhuman : atomsatoms more closer to Zincmore closer to Zinc (average (average - - 0.050.05 Å)Å)

HOH263-Zinc distance: effect on the fit

Human CA (A) ; Icefish CA (B)

Structure of the reaction centersStructure of the reaction centers

Coordinated waterCoordinated water: 1)1) CAiceCAice ( > Zn-OH distance):( > Zn-OH distance): >> higher pKa>> higher pKa

>> lower nuclephilicity>> lower nuclephilicity

pH-bond network:pH-bond network: 2)2) CAice (HOH263 and Thr199 closer and shifted consistently)

>> first H-bonding position more distant to the Metal>> first H-bonding position more distant to the Metal

Blue = CAiceBlue = CAice

Red = CA2hRed = CA2h

Zn2+

Thr199

HOH263

Template for modelling : 2cba

Modelling with SwissModel/DeepView3.7 and MOE

Homology modelingHomology modeling

N-term:

1) 2cba has lower resolution (higher uncertainty on first 30 positions)

2) Lower conservation between 2cba and CAice

Validation:

SAVS ( www.doe-mbi.ucla.edu/Servicies)

Protein report (MOE)

Score finale (Errat): 95,600 (100 max teorico)

Surface Electrostatic Potential distribution V(S)Surface Electrostatic Potential distribution V(S)

Extimated values (Hex4.5) (Hex4.5) : in vacuo assumption

CA2hCA2h: V=: V= +0.62 mV CAice : V= : V= -0.23 mV >> Icefish>> Icefish: : 1) negative potential1) negative potential

2) high number of net-charged residues in surface proximity

CA2h CAiceentrance to the enzymatic cleft

CA2hCAice

Surface Electrostatic Potential distribution V(S)Surface Electrostatic Potential distribution V(S)

>> Icefish:>> Icefish: negative potential around the entrance to the enzymatic cleft negative potential around the entrance to the enzymatic cleft

Surface potential distribution V(S) : Icefish peculiarity?

icefish

Onchorinchus mykiss

Tribolodon hakonensis Danio rerio

C.hamatus

Icefish (-0,23 mV)

Onchorinchus (+0,60 mV)

Tribolodon (+0,34 mV)

Zebrafish (+0,49 mV)

>> Negative V(S): Icefish peculiarity Net formal charge -7(+3 average for CA fish)

Considering the 100% aacidic identity in RC:

>> Control on the enzymatic activity: selective pressure on extra-RC positions for

chemical-physical properties distribution?

Temperature adaptation

?

Structural effects on the active site

Surface electrostatic potential

different kinetic parameters between CAice and CA2hNegative Potential:Negative Potential:Icefish peculiarityIcefish peculiarity

Sequence analysis

+

1) 1) ~~100 % conservation RC100 % conservation RC

2) Non conservative mutations:2) Non conservative mutations: beyond 15 A from Zincbeyond 15 A from Zinc

>> control on CA enzymatic activity: selective pressure on extra-RC positions

(i.e. Icefish : for precise chemical-physical properties distribution?)

Aknowledgements:

We thank Dr. I. Ascone for the excellent support at the LURE facility