International School for Advanced Studies
Metallo b-lactamases:
substrate binding and
catalytic mechanism
Matteo Dal Peraro
b-Lactam antibiotics
1928
Fleming
Bacterial
resistance
Bacterial resistance
 300 bacterial b-lactamases
• Serine hydrolases (classes A, C, D)
• Metallo hydrolases (class B)
 10%of b-lactamases
broad spectrum
no clinically useful inhibitors!
Zinc b-lactamases (MBL)
BcII
CcrA
IMP-1
VIM-2
BlaB
IND-1
Bacillus cereus
Bacteroides fragilis
Serratia marcescens, P. aeruginosa
Pseudomonas aeruginosa
Chryseobacterium meningosepticum
Chryseobacterium indologenes
B2
CphA
Sfh-I
imiS
Aeromonas hydrophila
Serratia fonticola
Aeromonas veronii bv. sobria
B3
L1
FEZ-1
GOB-1
Stenotrophomonas maltophilia
Fluoribacter goormanii
Chryseobacterium meningosepticum
B1
B1 MBL fold
L12
L3
Zn
N
C
Active species depend on bacterial strains
His263
Cys221
OH
Arg121
Asp120
His196
Zn1 His118
WAT
OH
Zn2
Zn1
His116
MONOZINC
B.cereus BcII
BIZINC
B.fragilis CcrA
Outlook
1. Monozinc BcII
2. Bizinc CcrA
• Several methodologies
• Feedback with experiments
(Vila Biophysics Lab)
 Second shell ligands
(Gervasio et al. 2003)
BcII monozinc hydration pattern
His196
Cys221
His263
Diaz et al., JACS 2001
OH
His118
Zn1
Rasia & Vila,
Biochemistry 2002
Arg121
Asp120
His116
M. Dal Peraro, A.J. Vila and P. Carloni, JBIC, 2002
Hydrolysis mechanism
MBL
OH-Zn
proton shuttle
No available inhibitor-enzyme complexes
MD structural model
Cefotaxime
(CEF, cephalosporin)
BcII-CEF binding mechanism
AMBER
•Active site
•CEF
L3
CEF
L12
C
Zn
N
M. Dal Peraro, A.J. Vila and P. Carloni, Proteins: SFG 2003
Asn233
Lys224
CEF
WAT
OH
His263
Zn1
Cys221
His118
Asp120
His116
Arg121
L12
Asn233
Zn-OH
L3
Lys224
CEF
L12
CEF
Asn233
Lys224
L3
ES
CEF
His263
WAT
OH
Zn
Cys221
Asp120
Arg121
His196
His118
His116
TS1
CEF
18.5 kcal/mol
His263
WAT
OH
Zn
Cys221
Asp120
Arg121
His196
His118
His116
+H2O
TS1 gas phase models
<
10 kcal/mol
Kleifeld, et al NSB 2003
INT
CEF
His263
His196
WAT
Cys221
Arg121
Zn
Asp120
His118
His116
TS2
21 kcal/mol
CEF*
WAT’
His196
WAT
His263
Cys221
Zn
His118
Asp120
Arg121
His116
EP
CEF*
His196
His263
WAT’
OH’
Cys221
Zn
Asp120
Arg121
His116
His118
Is the water-assisted mechanism
a common feature?
Benzylpenicillin
(BPC, penicillin)
-
Imipenem
(IMI, carbapenem)
-
Summary
• Water-assisted mechanism
• Asp120
• Arg121, Cys221, His263
Asp120 mutants
WT
D120E
D120N
D120S
k cat (s -1)
43(2)
5.1(7)
1.2(1)
4.2 (2)*10 -3
K M ( m M)
9(1)
110(20)
139(15)
45(7)
k cat /K M (M -1. s -1)
4.9(2)*10 6
4.6(15) *10 4
8.6(17) *10 3
93(19)
CcrA bizinc hydration pattern
Diaz et al., JACS 2001
M. Dal Peraro, A.J. Vila and P. Carloni, Inorg. Chem. 2003
CcrA binding mode
Asn233
Lys224
CEF
WAT
His263
His118
Zn2 OH
Zn1
Cys221
Asp120
His116
CcrA binding mode
Functional equivalence
Zn2-Asp120 bizinc
Asp120-Arg121 monozinc
BcII
CcrA
Conclusions
• Importance of outer shell ligands
• Water-assisted mechanism
• Role of aminoacids in the pocket (Asp120)
• General functional pattern
• Rational inhibitors design in Vila Lab
Acknowledgments
Prof. Paolo Carloni
Prof. Alejandro J. Vila, University Rosario
INFM, Democritos Center
HHMI, Howard Hughes Medical Institute
Acknowledgments 2
All the beautiful persons known in these years at SISSA
In particular, Michele, Pietro, Katrin
Computational Details
Geometry Optimization
Force Field Molecular Dynamics
•CPMD code, version 3.4 (Hutter et al, 2000)
• Direct Inversion in the Iteractive Subspace
(DIIS) algorithm (Pulay et al, 1984).
• Isolated System Method
(Martyna & Tuckerman, 1999).
• BLYP Exchange-Correlation Functional
(Becke, 1988; Lee,Yang & Parr, 1988).
• Plane Waves Cutoff Energy: 70 Ry.
• Norm Conserving Troullier-Martins PPs.
• Convergence: 5*E-4 GNMax.
•Amber 6 code (Case et al, UCSF)
• Amber force field (parm98.dat)
• Resp parameterization of substrate
and active site.
• Timestep 1 fs
• NPT ensemble, 298 K
• Particle Mesh Ewald
• SHAKE (omitted H-X bond interactions)
• Atoms: ~ 28.000
Energetics
•Energy Correction [DH(H+)]
to compare different protomers.
• D H(H+) estimation by waters clusters
optimizations: D H(H+)  250 kcal/mol
(exp. 263 kcal/mol)
(see Dal Peraro et al JBIC 2002).
QM-MM Hybrid Dynamics
•CPMD code, QM/MM version (Laio et al. 2002)
• BLYP Exchange-Correlation Functional
• Plane Waves Cutoff Energy: 70 Ry.
• Norm Conserving Troullier-Martins PPs.
• Timestep 6 a.u.
• Electronic Mass 900 a.u.
• Nose’-Hoover Thermostat, 298 K
• Constrained dynamics (Ciccotti et al. 1989)
• Quantum atoms: 52 on 28.000.
CP QM/MM dynamics
(Laio et al. 2002)
HTOT  Tel  Tion  EQM  EMM  EQM / MM
EQM  EKS
bonded
no bonded
EMM  EMM
 EMM
QM
r(r)
el
EQM
/ MM   qi
iIR

QM
 q Q
i
iMM
qi

r  r  vi eff  r  ri  dr 
vi eff  r  ri
QM
 DQM  QM  ...

MM
r
FEZ-1+cefotaxime
bizinc B3
CEF
Asn233
Ser228
His118
WAT
His263
OH
Zn2
Zn1
His121
Asp120
His116
BcII+benzylpenicillin
monozinc B1
Asn233
BPC
Lys224
His263
WAT
OH
Zn
Cys221
His118
Asp120
His116
Arg121
BcII+imipenem
monozinc B1
Asn233
IMI
Lys224
WAT
His263
OH
Cys221
Asp120
Zn
His118
His116
Arg121
CcrA+cefotaxime
bizinc B1
Asn233
Lys224
CEF
WAT
His263
His118
Zn2 OH
Zn1
Cys221
Asp120
His116
Hydrolysis mechanism
Cefotaxime (CEF) is a potent latest-generation cephalosporin
it is hydrolized by zinc-b-lactamases
(WT BcII : kcat/kM = 8.2 * 105)
BcII
CEF
(monozinc B1)
+
ES
TS1
nucleophilic attack
INT
TS2
N-C breaking
EP
Bizinc Hydration Pattern
Asp196
WAT
His263
Zn2
Cys221
Pro53(bb)
Asp120
His196
OH
Zn1
His118
His116
Suarez et al.,
Biochemistry 2002
Asn98
M. Dal Peraro, A.J. Vila and P. Carloni, Inorg Chem. 2003, 42:4245
Free energy profile
[email protected]
Scarica

Asp120 - Sissa