Mutations in Streptococcus pneumoniae penicillin-binding protein 2x: importance of the C-terminal penicillin-binding protein and serine/threonine kinase-associated domains for beta-lactam binding

Microb Drug Resist. 2012 Jun;18(3):314-21. doi: 10.1089/mdr.2012.0022. Epub 2012 Mar 28.

Abstract

Penicillin-binding protein 2x (PBP2x) mutations that occur during the selection with beta-lactams are located within the central penicillin-binding/transpeptidase (TP) domain, and are believed to mediate resistance by interfering with the formation of a covalent complex of the active site serine with the antibiotic. We now investigated the effect of two point mutations found in two independently obtained laboratory mutants that are located at the surface of the TP domain with their side chains facing outside (G422D respectively R426C). They have no significant effect on resistance to cefotaxime in vivo or on binding to Bocillin™FL to the active site in vitro using purified PBP2x derivatives, thus apparently do not affect the active site directly. In contrast, in silico modeling revealed that they affect van der Waal's interactions with the PASTA1 (PBP and serine/threonine kinase associated) domain of the C-terminal extension and a noncovalent cefuroxime molecule found in the X-ray structure of an acylated PBP2x, suggesting some effect of the mutations on the interaction of the TP domain with PASTA1 and/or with the antibiotic associated with PASTA1. The effect of the PASTA domains on covalent binding of PBP2x to Bocillin FL was then investigated using a series of soluble truncated PBP2x derivatives. Deletion of 127 C-terminal residues, that is, of both PASTA domains, decreased binding dramatically by ∼90%. Surprisingly, deletion of only 40 amino acids resulted in the same phenotype, whereas the absence of 30 amino acids affected binding marginally by 10%, documenting a crucial role of the C-terminal domain for beta-lactam binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / chemistry
  • Anti-Bacterial Agents / metabolism*
  • Binding Sites
  • Boron Compounds / chemistry
  • Boron Compounds / metabolism
  • Cefotaxime / chemistry
  • Cefotaxime / metabolism*
  • Cefuroxime / chemistry
  • Cefuroxime / metabolism*
  • Computer Simulation
  • Crystallography, X-Ray
  • Drug Resistance, Microbial / genetics*
  • Models, Molecular
  • Penicillin-Binding Proteins / chemistry
  • Penicillin-Binding Proteins / genetics*
  • Penicillin-Binding Proteins / metabolism
  • Penicillins / chemistry
  • Penicillins / metabolism
  • Point Mutation
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Serine / chemistry
  • Serine / metabolism
  • Streptococcus pneumoniae / genetics*
  • Streptococcus pneumoniae / metabolism
  • Structure-Activity Relationship

Substances

  • Anti-Bacterial Agents
  • BOCILLIN FL
  • Boron Compounds
  • Penicillin-Binding Proteins
  • Penicillins
  • Recombinant Proteins
  • PBP 2x protein, Streptococcus
  • Serine
  • Protein-Serine-Threonine Kinases
  • Cefotaxime
  • Cefuroxime