Crystal Structures of Penicillin-binding Protein 2 from Penicillin-susceptible and -resistant Strains of Neisseria gonorrhoeae Reveal an Unexpectedly Subtle Mechanism for Antibiotic Resistance

Abstract

Penicillin-binding protein 2 (PBP2) from N. gonorrhoeae is the major molecular target for ␤-lactam antibiotics used to treat gonococcal infections. PBP2 from penicillin-resistant strains of N. gonorrhoeae harbors an aspartate insertion after position 345 (Asp-345a) and 4 -8 additional mutations, but how these alter the architecture of the protein is unknown. We have determined the crystal structure of PBP2 derived from the penicillin-susceptible strain FA19, which shows that the likely effect of Asp-345a is to alter a hydrogen-bonding network involving Asp-346 and the SXN triad at the active site. We have also solved the crystal structure of PBP2 derived from the penicillin-resistant strain FA6140 that contains four mutations near the C terminus of the protein. Although these mutations lower the second order rate of acylation for penicillin by 5-fold relative to wild type, comparison of the two structures shows only minor structural differences, with the positions of the conserved residues in the active site essentially the same in both. Kinetic analyses indicate that two mutations, P551S and F504L, are mainly responsible for the decrease in acylation rate. Melting curves show that the four mutations lower the thermal stability of the enzyme. Overall, these data suggest that the molecular mechanism underlying antibiotic resistance contributed by the four mutations is subtle and involves a small but measurable disordering of residues in the active site region that either restricts the binding of antibiotic or impedes conformational changes that are required for acylation by ␤-lactam antibiotics.Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection, gonorrhea, is an obligate human pathogen that primarily colonizes the urogenital tract. For nearly 40 years N. gonorrhoeae was treated with a single dose of penicillin, but in 1987 the prevalence of penicillin-resistant strains necessitated the use of alternative antibiotics (1), primarily fluoroquinolones and third-generation cephalosporins. The rapid emergence and dissemination of fluoroquinolone-resistant N. gonorrhoeae, however, leaves only third-generation cephalosporins (e.g. cefixime and ceftriaxone) as recommended agents (1). Even though these cephalosporins are still effective anti-gonococcal antibiotics, the increasing prevalence of strains with intermediate resistance to these agents suggests that full resistance is not far behind (2-5). These trends signal an urgent need to understand the molecular mechanisms of antibiotic resistance and to devise new treatment options for gonococcal infections.The molecular targets of ␤-lactam antibiotics are the penicillin-binding proteins (PBPs), 3 a group of enzymes involved in the final stages of peptidoglycan synthesis in bacteria (6 -8). PBPs are grouped into three main classes, A, B, and C. Class A and B PBPs are transpeptidases (TPases) that catalyze the formation of peptide cross-links between adjacent glycan strands of peptidoglycan, but are distinguished in that class A PBPs also conta...

Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape

“…We considered the enzymes' crystal structures for a clue to how the first crosslink might be formed on a new strand. While the transpeptidase active site must be positioned farther into the periplasm to interact with the sacculus, the transglycosylase active site, being next to the transmembrane region, must be near the inner membrane (55,60,61). Thus, newly added disaccharides must move up from the inner membrane to the sacculus before they can be crosslinked, presumably passing directly from the transglycosylase to the transpeptidase active sites (55).…”

Identification of Amino Acids Conferring High-Level Resistance to Expanded-Spectrum Cephalosporins in the penA Gene from Neisseria gonorrhoeae Strain H041

“…PBP2 mutant proteins were purified and used to determine the k 2 /K s values for their acylation rates with penicillin, ceftriaxone, and cefixime, as described previously (25,31). The reaction of ␤-lactam antibiotics with PBP2 is denoted by the equation…”

“…k 2 /K s constants, which are a direct measure of the ability of an antibiotic to inhibit a PBP (32), were calculated from first-order rates of acylation of purified, soluble PBP2 variants by [ 14 C]penicillin G (Moravek, Brea, CA), as previously described (25,31,33 …”

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

“…The small adjacent N‐terminal segment of the transpeptidase domain comprises two α‐helices [α1n (250–268) and α2n (270–276)] and a three‐stranded [β1n (280–284), β2n (480–486), and β3n (490–495)] antiparallel β‐sheet (Figs 1A and 2). The PonA1 transpeptidase (or penicillin‐binding domain) resembles other PBPs secondary structure: PBP1 of class A from Staphylococcus pneumonia (RMSD of 1.9 Å) 24, 25, the transpeptidase domain of PBP2 from Neisseria gonorroeae (RMSD of 2.00 Å) 26, class A PBP1 from Pseudomonas aeruginosa (RMSD of 1.65 Å) 27, 28, and PBP4 from Listeria monocytogenes (RMSD of 2.01 Å) 29. A cleft in the middle of the domain contains the active site.…”