Structural basis for the β lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus

The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad-spectrum β-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Because of its low affinity for β-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at β-lactam concentrations that inhibit the β-lactam-sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 Å resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefin, penicillin G and methicillin allow, for the first time, a comparison of an apo and acylated resistant PBP. An analysis of the PBP2a active site in these forms reveals the structural basis of its resistance and identifies features in newly developed β-lactams that are likely important for high affinity binding.

[1]  A. Tomasz,et al.  Abnormal Peptidoglycan Produced in a Methicillin-Resistant Strain of Staphylococcus aureus Grown in the Presence of Methicillin: Functional Role for Penicillin-Binding Protein 2A in Cell Wall Synthesis , 1993, Antimicrobial Agents and Chemotherapy.

[2]  M. Page,et al.  Crystal structure of the class D β-lactamase OXA-10 , 2000, Nature Structural Biology.

[3]  P. Reynolds,et al.  Penicillin‐binding proteins of β‐lactam‐resistant strains of Staphylococcus aureus , 1985, FEBS letters.

[4]  R Miller,et al.  Optimizing Shake-and-Bake for proteins. , 1999, Acta crystallographica. Section D, Biological crystallography.

[5]  J. Frère,et al.  Active-site-serine D-alanyl-D-alanine-cleaving-peptidase-catalysed acyl-transfer reactions. Procedures for studying the penicillin-binding proteins of bacterial plasma membranes. , 1986, The Biochemical journal.

[6]  W. Hendrickson.  Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. , 1991, Science.

[7]  R. Read.  Improved Fourier Coefficients for Maps Using Phases from Partial Structures with Errors , 1986 .

[8]  I. Medina,et al.  In Vitro and In Vivo Activities of a Novel Cephalosporin, BMS-247243, against Methicillin-Resistant and -Susceptible Staphylococci , 2002, Antimicrobial Agents and Chemotherapy.

[9]  Michael F. Summers,et al.  Solution Structure of 3-Oxo-Δ5-Steroid Isomerase , 1997 .

[10]  R. Brasseur,et al.  The Crystal Structure of a Penicilloyl-serine Transferase of Intermediate Penicillin Sensitivity , 1999, The Journal of Biological Chemistry.

[11]  J. Hermes,et al.  High-yield expression, refolding, and purification of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus strain 27R. , 1995, Protein expression and purification.

[12]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[13]  G. Basarab,et al.  Structure-based design of potent inhibitors of scytalone dehydratase: displacement of a water molecule from the active site. , 1998, Biochemistry.

[14]  O. Dideberg,et al.  The crystal structure of the penicillin-binding protein 2x from Streptococcus pneumoniae and its acyl-enzyme form: implication in drug resistance. , 2000, Journal of molecular biology.

[15]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[16]  T. Vernet,et al.  Identification of a structural determinant for resistance to beta-lactam antibiotics in Gram-positive bacteria. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Kanehisa,et al.  Whole genome sequencing of meticillin-resistant Staphylococcus aureus , 2001, The Lancet.

[18]  J. Frère,et al.  Kinetics of interaction between the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61 and beta-lactam antibiotics. A choice of models. , 1975, European journal of biochemistry.

[19]  J. Ghuysen,et al.  Multimodular Penicillin-Binding Proteins: An Enigmatic Family of Orthologs and Paralogs , 1998, Microbiology and Molecular Biology Reviews.

[20]  R N Jones,et al.  Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997-1999. , 2001, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[21]  R. Pratt,et al.  Reaction of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with beta-lactams and acyclic substrates: kinetics in homogeneous solution. , 1998, The Biochemical journal.

[22]  C. Betzel,et al.  Molecular structure of the acyl-enzyme intermediate in β-lactam hydrolysis at 1.7 Å resolution , 1992, Nature.

[23]  K. Hiramatsu.  Molecular Evolution of MRSA , 1995, Microbiology and immunology.

[24]  J M Ghuysen,et al.  2.8-A Structure of penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase from Streptomyces R61 and complexes with beta-lactams. , 1986, The Journal of biological chemistry.

[25]  Thomas C. Terwilliger,et al.  Electronic Reprint Biological Crystallography Maximum-likelihood Density Modification , 2022 .

[26]  Y. Fukuchi,et al.  Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin , 1997, The Lancet.

[27]  K. Hiramatsu,et al.  Eagle-Type Methicillin Resistance: New Phenotype of High Methicillin Resistance under mec Regulator Gene Control , 2001, Antimicrobial Agents and Chemotherapy.

[28]  P. Skatrud,et al.  Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate , 1992, Antimicrobial Agents and Chemotherapy.

[29]  A. Tomasz,et al.  Complementation of the Essential Peptidoglycan Transpeptidase Function of Penicillin-Binding Protein 2 (PBP2) by the Drug Resistance Protein PBP2A in Staphylococcus aureus , 2001, Journal of bacteriology.

[30]  A. Tomasz,et al.  Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus , 1984, Journal of bacteriology.

[31]  Y. Lindqvist,et al.  Crystal structure of scytalone dehydratase--a disease determinant of the rice pathogen, Magnaporthe grisea. , 1994, Structure.

[32]  B. Leiting,et al.  Soluble Penicillin-Binding Protein 2a: β-Lactam Binding and Inhibition by Non-β-Lactams Using a 96-Well Format , 1998 .

[33]  G. Bricogne,et al.  [27] Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. , 1997, Methods in enzymology.

[34]  O. Dideberg,et al.  Crystal Structure of PBP2x from a Highly Penicillin-resistant Streptococcus pneumoniae Clinical Isolate , 2001, The Journal of Biological Chemistry.

[35]  H. Chambers.  Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications , 1997, Clinical microbiology reviews.

[36]  C. Sander,et al.  Protein structure comparison by alignment of distance matrices. , 1993, Journal of molecular biology.

[37]  G. Cohen.  Align : A program to superimpose protein coordinates, accounting for insertions and deletions , 1997 .

[38]  R. Krishnaraj,et al.  Effect of side-chain amide thionation on turnover of beta-lactam substrates by beta-lactamases. Further evidence on the question of side-chain hydrogen-bonding in catalysis. , 1992, The Biochemical journal.

[39]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[40]  M. Kuroda,et al.  The emergence and evolution of methicillin-resistant Staphylococcus aureus. , 2001, Trends in microbiology.

[41]  W. Yeh,et al.  Purification, properties, and kinetics of enzymatic acylation with beta-lactams of soluble penicillin-binding protein 2a. A major factor in methicillin-resistant Staphylococcus aureus. , 1994, The Journal of biological chemistry.

[42]  O. Dideberg,et al.  X-ray structure of Streptococcus pneumoniae PBP2x, a primary penicillin target enzyme , 1996, Nature Structural Biology.

[43]  I. Massova,et al.  Kinship and Diversification of Bacterial Penicillin-Binding Proteins and β-Lactamases , 1998, Antimicrobial Agents and Chemotherapy.

[44]  S. White,et al.  Crystal Structure of a Deacylation-defective Mutant of Penicillin-binding Protein 5 at 2.3-Å Resolution* , 2001, The Journal of Biological Chemistry.

[45]  E. Sauvage,et al.  The 2.4-Å crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin , 2002, Cellular and Molecular Life Sciences CMLS.

[46]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[47]  Y. Sun,et al.  Kinetics of beta-lactam interactions with penicillin-susceptible and -resistant penicillin-binding protein 2x proteins from Streptococcus pneumoniae. Involvement of acylation and deacylation in beta-lactam resistance. , 2001, The Journal of biological chemistry.

[48]  J M Ghuysen,et al.  Penicillin-binding proteins. Wall peptidoglycan assembly and resistance to penicillin: facts, doubts and hopes. , 1997, International journal of antimicrobial agents.

[49]  D. McRee,et al.  A visual protein crystallographic software system for X11/Xview , 1992 .

[50]  M. Ishiguro,et al.  Modeling study on a hydrolytic mechanism of class A β-lactamases , 1996 .

[51]  Y. Sun,et al.  Penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: kinetic characterization of its interactions with beta-lactams using electrospray mass spectrometry. , 1999, Biochemistry.

[52]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[53]  E. Conti,et al.  Structural basis for the recognition of a nucleoporin FG repeat by the NTF2-like domain of the TAP/p15 mRNA nuclear export factor. , 2001, Molecular cell.

[54]  P. Moreillon,et al.  BAL9141, a Novel Extended-Spectrum Cephalosporin Active against Methicillin-Resistant Staphylococcus aureus in Treatment of Experimental Endocarditis , 2002, Antimicrobial Agents and Chemotherapy.