The crystal structure of the penicillin-binding protein 2x from Streptococcus pneumoniae and its acyl-enzyme form: implication in drug resistance.

Penicillin-binding proteins (PBPs), the primary targets for beta-lactam antibiotics, are periplasmic membrane-attached proteins responsible for the construction and maintenance of the bacterial cell wall. Bacteria have developed several mechanisms of resistance, one of which is the mutation of the target enzymes to reduce their affinity for beta-lactam antibiotics. Here, we describe the structure of PBP2x from Streptococcus pneumoniae determined to 2.4 A. In addition, we also describe the PBP2x structure in complex with cefuroxime, a therapeutically relevant antibiotic, at 2.8 A. Surprisingly, two antibiotic molecules are observed: one as a covalent complex with the active-site serine residue, and a second one between the C-terminal and the transpeptidase domains. The structure of PBP2x reveals an active site similar to those of the class A beta-lactamases, albeit with an absence of unambiguous deacylation machinery. The structure highlights a few amino acid residues, namely Thr338, Thr550 and Gln552, which are directly related to the resistance phenomenon.

[1]  A. Brünger,et al.  Torsion angle dynamics: Reduced variable conformational sampling enhances crystallographic structure refinement , 1994, Proteins.

[2]  A. Brunger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. , 1992 .

[3]  J. Frère,et al.  Penicillin binding protein 2x as a major contributor to intrinsic β‐lactam resistance of Streptococcus pneumoniae , 1993, FEBS letters.

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

[5]  A. Kuzin,et al.  Binding of cephalothin and cefotaxime to D-ala-D-ala-peptidase reveals a functional basis of a natural mutation in a low-affinity penicillin-binding protein and in extended-spectrum beta-lactamases. , 1995, Biochemistry.

[6]  J. Frère,et al.  Catalytic properties of class A beta-lactamases: efficiency and diversity. , 1998, The Biochemical journal.

[7]  Peter S. Shenkin,et al.  Amino Acid Sequence Determinants of β-Lactamase Structure and Activity , 1996 .

[8]  L. Gutmann,et al.  Acquisition of Five High-MrPenicillin-Binding Protein Variants during Transfer of High-Level β-Lactam Resistance from Streptococcus mitis toStreptococcus pneumoniae , 1998, Journal of bacteriology.

[9]  B. Spratt,et al.  Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae. , 1993, FEMS microbiology letters.

[10]  G. Becker,et al.  Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics , 1997, Journal of bacteriology.

[11]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[12]  K Cowtan,et al.  Miscellaneous algorithms for density modification. , 1998, Acta crystallographica. Section D, Biological crystallography.

[13]  J. Frère,et al.  The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[14]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

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

[16]  G. Laible,et al.  Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae , 1991, Journal of bacteriology.

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

[18]  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.

[19]  J. Höltje,et al.  Growth of the Stress-Bearing and Shape-Maintaining Murein Sacculus of Escherichia coli , 1998, Microbiology and Molecular Biology Reviews.

[20]  O. Massidda,et al.  Unconventional organization of the division and cell wall gene cluster of Streptococcus pneumoniae. , 1998, Microbiology.

[21]  J. Frère,et al.  Mechanism of acyl transfer by the class A serine beta-lactamase of Streptomyces albus G. , 1991, The Biochemical journal.

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

[23]  J. Frère,et al.  Site-directed mutagenesis of glutamate 166 in two beta-lactamases. Kinetic and molecular modeling studies. , 1997, The Journal of biological chemistry.

[24]  R. Hakenbeck,et al.  Penicillin‐binding proteins in β‐lactam‐resistant laboratory mutants of Streptococcus pneumoniae , 1987, Molecular microbiology.

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

[26]  A. Tomasz,et al.  Separation of abnormal cell wall composition from penicillin resistance through genetic transformation of Streptococcus pneumoniae , 1996, Journal of bacteriology.

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

[28]  R. Huber,et al.  Accurate Bond and Angle Parameters for X-ray Protein Structure Refinement , 1991 .

[29]  V S Lamzin,et al.  Automated refinement of protein models. , 1993, Acta crystallographica. Section D, Biological crystallography.

[30]  Laurent Maveyraud,et al.  Crystal Structure of 6Alpha-Hydroxymethylpenicillanate Complexed to the Tem-1 Beta-Lactamase from Escherichia Coli: Evidence on the Mechanism of Action of a Novel Inhibitor Designed by a Computer-Aided Process , 1996 .

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

[32]  R. Hakenbeck,et al.  Penicillin-binding proteins 2b and 2x of Streptococcus pneumoniae are primary resistance determinants for different classes of beta-lactam antibiotics , 1996, Antimicrobial agents and chemotherapy.

[33]  Axel T. Brunger,et al.  X-PLOR Version 3.1: A System for X-ray Crystallography and NMR , 1992 .

[34]  J M Thornton,et al.  LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. , 1995, Protein engineering.

[35]  F Jacob,et al.  Role of the conserved amino acids of the 'SDN' loop (Ser130, Asp131 and Asn132) in a class A beta-lactamase studied by site-directed mutagenesis. , 1990, The Biochemical journal.

[36]  T. Vernet,et al.  Mutations in the Active Site of Penicillin-binding Protein PBP2x from Streptococcus pneumoniae , 1999, The Journal of Biological Chemistry.

[37]  R. Wade,et al.  pKa Calculations for class A β‐lactamases: Influence of substrate binding , 2008 .

[38]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[39]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[40]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[41]  L. Gutmann,et al.  In vitro selection of one-step mutants of Streptococcus pneumoniae resistant to different oral beta-lactam antibiotics is associated with alterations of PBP2x , 1996, Antimicrobial agents and chemotherapy.

[42]  J M Masson,et al.  Crystal structure of Escherichia coli TEM1 β‐lactamase at 1.8 Å resolution , 1993, Proteins.

[43]  K. Ubukata,et al.  Diversity of Substitutions within or Adjacent to Conserved Amino Acid Motifs of Penicillin-Binding Protein 2X in Cephalosporin-Resistant Streptococcus pneumoniaeIsolates , 1999, Antimicrobial Agents and Chemotherapy.