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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