Conformational Dynamics in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus, Allosteric Communication Network and Enablement of Catalysis

Abstract

The mechanism of the β-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism. An enzyme, called penicillin-binding protein 2a (PBP2a), is brought into this biosynthetic pathway to complete the cross-linking. PBP2a effectively discriminates against the β-lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site, and realigns the conformation of key residues to permit catalysis. We address the molecular basis of this regulation using crystallographic studies augmented by computational analyses. The crystal structures of three β-lactams (oxacillin, cefepime, ceftazidime) complexes with PBP2a—each with the β-lactam in the allosteric site—defined (with preceding PBP2a structures) the “open” or “partially open” PBP2a states. A particular loop motion adjacent to the active site is identified as the driving force for the active-site conformational change that accompanies active-site opening. Correlation of this loop motion to effector binding at the allosteric site, in order to identify the signaling pathway, was accomplished computationally in reference to the known “closed” apo-PBP2a X-ray crystal structure state. This correlation enabled the computational simulation of the structures coinciding with initial peptidoglycan substrate binding to PBP2a; acyl-enzyme formation; and acyl-transfer to a second peptidoglycan substrate to attain cross-linking. These studies offer important insights into the structural bases for allosteric site to active site communication and for β-lactam mimicry of the peptidoglycan substrates, as foundational to the mechanistic understanding of emerging PBP2a resistance mutations.

Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa

“…S7A). The full occupancy complex for Slt was submitted to molecular-dynamics simulations for a duration of 200 ns according to the methodology reported previously (23) in the program AMBER16 program (24) (SI Materials and Methods). The simulation demonstrated a stable binding of the substrate within the extended substratebinding groove.…”

BLIP-II Employs Differential Hotspot Residues To Bind Structurally Similar Staphylococcus aureus PBP2a and Class A β-Lactamases

“…The dynamic nature and allosteric regulation of the transpeptidase domain of PBP2a has been established in crystal structures (29, 30) . This structural plasticity may have implications in the slow rate of association and overall weak binding of BLIP-II with PBP2a.…”

Constructing and deconstructing the bacterial cell wall

“…The stereoview of the active site of S. aureus PBP2a acylated by the cephalosporin, ceftaroline (PDB 3ZG0) . Ring opening of the β‐lactam requires catalysis by a lysine general base (K406) that activates acyl‐transfer from the β‐lactam to the nucleophilic serine (S403).…”

“…Following translocation, the allosteric site of PBP2a engages existing “template” peptidoglycan so as to trigger the conformational change that opens the active site. Only then is new peptidoglycan structure preferentially admitted for catalysis …”

“…Only then is new peptidoglycan structure preferentially admitted for catalysis. 141 The identification of this allosteric site had serendipitous consequence. Virtual screening using our PBP2a structure identified two classes of small molecule adjuvants that in murine pharmacological assay restored the antibiotic activity of β-lactams against S. aureus (as well as against other Grampositive pathogens).…”