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 iIR QM q Q i iMM 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 O@OH