International School for Advanced Studies

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International School for Advanced
Studies Metallo b-lactamases substrate
binding and catalytic mechanism Matteo Dal
Peraro
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b-Lactam antibiotics
1928 Fleming
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Bacterial resistance

• ? 300 bacterial b-lactamases
• Serine hydrolases (classes A, C, D)
• Metallo hydrolases (class B)
• ? 10of b-lactamases

?broad spectrum ?no clinically useful inhibitors!
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Zinc b-lactamases (MBL)
BcII Bacillus cereus CcrA Bacteroides
fragilis IMP-1 Serratia marcescens, P.
aeruginosa VIM-2 Pseudomonas aeruginosa
BlaB Chryseobacterium meningosepticum IND-1 Chr
yseobacterium indologenes CphA Aeromonas
hydrophila Sfh-I Serratia fonticola imiS Aeromon
as veronii bv. sobria L1 Stenotrophomonas
maltophilia FEZ-1 Fluoribacter
goormanii GOB-1 Chryseobacterium
meningosepticum
B1
B2
B3
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B1 MBL fold
L12
L3
C
Zn
N
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Active species depend on bacterial strains
His196
WAT
His263
Cys221
OH
OH
His118
Zn1
Zn2
Zn1
Asp120
Arg121
His116
BIZINC B.fragilis CcrA
MONOZINC B.cereus BcII
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Outlook

• Monozinc BcII
• Bizinc CcrA
• Several methodologies
• Feedback with experiments
• (Vila Biophysics Lab)
• ? Second shell ligands
• (Gervasio et al. 2003)

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BcII monozinc hydration pattern
His196
Cys221
His263
His118
OH
Diaz et al., JACS 2001
Zn1
Asp120
Arg121
His116
M. Dal Peraro, A.J. Vila and P. Carloni, JBIC,
2002
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Hydrolysis mechanism
MBL
No available inhibitor-enzyme complexes
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MD structural model
Cefotaxime (CEF, cephalosporin)
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BcII-CEF binding mechanism
L12
CEF
L3

• AMBER
• Active site
• CEF

C
Zn
N
M. Dal Peraro, A.J. Vila and P. Carloni,
Proteins SFG 2003
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Asn233
Lys224
CEF
WAT
OH
His263
Zn1
His118
Cys221
Asp120
His116
Arg121
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ES
CEF
WAT
His263
His196
OH
Zn
Cys221
His118
Asp120
Arg121
His116
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TS1 18.5 kcal/mol
CEF
His263
His196
OH
WAT
Zn
Cys221
His118
Asp120
Arg121
His116
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H2O
lt ?10 kcal/mol
TS1 gas phase models
Kleifeld, et al NSB 2003
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INT
CEF
His263
His196
WAT
Cys221
Zn
His118
Asp120
Arg121
His116
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TS2 ?21 kcal/mol
CEF
His196
WAT
WAT
Fair agreemnt
His263
Zn
Cys221
His118
Asp120
His116
Arg121
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EP
CEF
His196
His263
WAT
OH
Zn
His118
Cys221
Asp120
Arg121
His116
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Is the water-assisted mechanism a common feature?
Benzylpenicillin (BPC, penicillin)
-
Imipenem (IMI, carbapenem)
-
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Summary

• Water-assisted mechanism
• Asp120
• Arg121, Cys221, His263

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Asp120 mutants

k
(s
)

m
k
/K
(M
. s
)

-
1
K
(
M)

-
1
-
1
cat
cat
M
M
WT

43(2)

9(1)

4.9
(2)
10


6
D120E

5.1
(7)

110(20)

4.6
(15)
10


4
D120N

1.2
(
1
)

139(15)

3
8.6
(17)
10

D
120S

4.2
(2)
10
45(7)

93(19)

-
3


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CcrA bizinc hydration pattern
Diaz et al., JACS 2001
M. Dal Peraro, A.J. Vila and P. Carloni, Inorg.
Chem. 2003
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CcrA binding mode
Equivalenza Asecons asfera con zn2
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CcrA binding mode
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Functional equivalence Zn2-Asp120
bizinc Asp120-Arg121 monozinc
Funct equi of 2 binding site Ruolo funt shell E
wat
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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

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Acknowledgments
Prof. Paolo Carloni Prof. Alejandro J. Vila,
University Rosario INFM, Democritos Center HHMI,
Howard Hughes Medical Institute
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Acknowledgments 2
All the beautiful persons known in these years at
SISSA In particular, Michele, Pietro, Katrin
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Computational Details

• Geometry Optimization
• 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 5E-4 GNMax.
• Energetics
• Energy Correction DH(H)
• to compare different protomers.
• D H(H) estimation by waters clusters

• Force Field Molecular Dynamics
• 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
• 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.

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CP QM/MM dynamics (Laio et al. 2002)
QM
r(r)
qi
MM
r
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FEZ-1cefotaxime bizinc B3
CEF
Asn233
Ser228
His118
WAT
OH
His263
Zn1
Zn2
His121
Asp120
His116
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BcIIbenzylpenicillin monozinc B1
Asn233
BPC
Lys224
WAT
OH
His263
Zn
His118
Cys221
Asp120
His116
Arg121
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BcIIimipenem monozinc B1
Asn233
IMI
Lys224
WAT
His263
OH
Zn
Cys221
His118
Asp120
His116
Arg121
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CcrAcefotaxime bizinc B1
Asn233
Lys224
CEF
His118
WAT
OH
Zn2
His263
Zn1
Cys221
His116
Asp120
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Hydrolysis mechanism
Cefotaxime (CEF) is a potent latest-generation
cephalosporin it is hydrolized by
zinc-b-lactamases (WT BcII kcat/kM 8.2
105)
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BcII (monozinc B1)
CEF

ES
TS1
INT
TS2
EP
N-C breaking
nucleophilic attack
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Bizinc Hydration Pattern
WAT
His196
His263
OH
Zn2
His118
Zn1
Cys221
His116
Asp120
Suarez et al., Biochemistry 2002
M. Dal Peraro, A.J. Vila and P. Carloni, Inorg
Chem. 2003, 424245
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Free energy profile
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O_at_OH