Crystal Structure of PBP2x from a Highly Penicillin-resistant Streptococcus pneumoniae Clinical Isolate

Abstract

Penicillin-binding proteins (PBPs) are the main targets for ␤-lactam antibiotics, such as penicillins and cephalosporins, in a wide range of bacterial species. In some Gram-positive strains, the surge of resistance to treatment with ␤-lactams is primarily the result of the proliferation of mosaic PBP-encoding genes, which encode novel proteins by recombination. PBP2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level ␤-lactam resistance. To understand such a resistance mechanism at an atomic level, we have solved the x-ray crystal structure of PBP2x from a highly penicillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* active site, the Thr 338 3 Ala mutation weakens the local hydrogen bonding network, thus abrogating the stabilization of a crucial buried water molecule. In addition, the Ser 389 3 Leu and Asn 514 3 His mutations produce a destabilizing effect that generates an "open" active site. It has been suggested that peptidoglycan substrates for ␤-lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains tend to catalyze cross-linking of linear forms. Thus, in vivo, an "open" active site could facilitate the recognition of distinct, branched physiological substrates.

The 2.4-Å crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin

“…In contrast, strains showing vancomycin resistance are now assumed to synthesize a pentapeptide with an abnormal carboxyterminal unable to bind vancomycin. The syner- [24], resistance to b-lactams is a constitutive element of E. faecium and a few point mutations in PBP5fm can lead to very high levels of resistance.…”

Large ring 1,3-bridged 2-azetidinones: Experimental and theoretical studies

“…[55-57] PBP 2a from Staphylococcus aureus ATCC433000 [58,59], PBP 5fm from Enterococcus faecium [60], and PBP 2x from Streptococcus pneumoniae 5204 [61][62][63]. The peptidases (0.8 or 2.5 mM) and the azetidinones 10-12 (100 mM) were incubated during 16 h, then fluorescent ampicillin (25 mM) was added.…”

Crystallization and initial X-ray diffraction study of the three PASTA domains of the Ser/Thr kinase Stk1 from the human pathogenStaphylococcus aureus

“…The XDS program package was used for data reduction and scaling (Table 1). Molecular replacement was attempted with the program AMoRe (Navaza, 2001) using the PASTA subunits of the 2.0 Å structure of PBP2X (Dessen et al, 2001) as search models. Every possible case was tested in terms of body search, assuming that our crystals may contain one or two molecules in the asymmetric unit.…”

“…To date, only one representative structure containing PASTA subunits is available in the Protein Data Bank (PDB): that of the high-molecular-mass type II penicillin-binding protein PBP2X from S. pneumoniae (PDB code 1k25; Dessen et al, 2001). PBP2X contains two PASTA subunits, each of which is composed of a small globular fold consisting of three -strands and an -helix.…”