How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function

Abstract

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain--a remarkable 60 Å distant from the DD-transpeptidase active site--discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.

Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B

“…Such long-range allosteric effects on PBP activity are not without precedent. Recently, the binding of the antibiotic ceftaroline or PG fragments to an allosteric site was shown to open the catalytic TPase site of PBP2a from a methicillin-resistant Staphylococcus aureus strain over a distance of ∼60 Å (29). Further experiments are required to determine exactly how LpoB stimulates PBP1B.…”

Targeting Antibiotic Resistance

“…For ceftaroline, an allosteric binding site at PBP2a has been identified by crystallographic analysis (Figure 32). The allosteric binding site (Figure 32 C) is separated from the active site (Figure 32 B) by a remarkable 60 Å distance (Figure 32 A) 148. Crystallographic analysis also revealed the identity of other allosteric ligands for PBP2, such as muramic acid (a saccharide component of the peptidoglycan).…”

“…Crystallographic analysis also revealed the identity of other allosteric ligands for PBP2, such as muramic acid (a saccharide component of the peptidoglycan). Therefore, it has been proposed that the function of the allosteric domain is to sense nascent peptidoglycan and then open the active site to catalyze the transpeptidation 148. The elucidation of the ability of the anti‐MRSA β‐lactam antibiotic ceftaroline and other molecules to trigger allosteric opening of the active site so that PBP2a can be inactivated by a second β‐lactam molecule, should enable future structure‐based design campaigns for β‐lactam antibiotics.…”

“…By kinetic studies and by X‐ray crystallography of the mutants, Mobashery, Hermoso et al. were able to rationalize that the clinically observed mutations interfere with triggering of the allosteric signal by ceftaroline along the interacting amino acid network between the two sites 148, 150. These mutations allow the mutant variants of PBP2a to manifest resistance to ceftaroline by two different mechanisms: the first is a modest increase in the dissociation constant for ceftaroline binding to the allosteric site, and the second is disruption of the transmission of conformational changes necessary for opening the active site.…”

Oxacillin magnetically targeted for the treatment of Methicillin-Resistant S. aureus infection in rats

“…These antibiotics acting by linkage with the penicillin binding proteins (PBPs), resulting in the inhibition of the cell wall synthesis and consequently growth inhibition or cell lysis. MRSA strains synthetize PBP2a, a protein of low affinity to bond β-lactams, therefore the cell wall can be renewed 14 . A range of new agents for the treatment of MRSA infections have resulted favorably, but usage of antibiotics has enhanced the accumulation of genetic elements coding for resistance, making effective therapies a current challenging goal 15,16 .…”