The Tipper-Strominger Hypothesis and Triggering of Allostery in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus (MRSA)

Abstract

The transpeptidases involved in the synthesis of the bacterial cell wall (also known as penicillin-binding proteins, PBPs) have evolved to bind the acyl-D-Ala-D-Ala segment of the stem peptide of the nascent peptidoglycan for the physiologically important cross-linking of the cell wall. The Tipper-Strominger hypothesis stipulates that β-lactam antibiotics mimic the acyl-D-Ala-D-Ala moiety of the stem and, thus, are recognized by the PBPs with bactericidal consequences. We document that this mimicry exists also at the allosteric site of PBP2a of methicillin-resistant Staphylococcus aureus (MRSA). Interactions of different classes of β-lactam antibiotics, as mimics of the acyl-D-Ala-D-Ala moiety at the allosteric site, lead to a conformational change, across a distance of 60 Å to the active site. We directly visualize this change using an environmentally sensitive fluorescent probe affixed to the protein loops that frame the active site. This conformational mobility, documented in real time, allows antibiotic access to the active site of PBP2a. Furthermore, we document that this allosteric trigger enables synergy between two different β-lactam antibiotics, wherein occupancy at the allosteric site by one facilitates occupancy by a second at the transpeptidase catalytic site, thus lowering the minimal-inhibitory concentration. This synergy has important implications for the mitigation of facile emergence of resistance to these antibiotics by MRSA.

Figure 1

The chemical structure of ceftaroline (as a representative cephalosporin) and its mimicry of the acyl-d-Ala-d-Ala terminus of the peptidoglycan pentapeptide (colored in blue) are shown. Chemical structures of a penicillin and a carbapenem are also shown. The mimicry is preserved across these three templates of the β-lactam family of antibiotics.

Figure 2

(A) The closed conformation of PBP2a with the side chains of Y446 and Q607 shown as capped sticks in black, indicated by arrows at 1 o’clock. (B) Reaction of the fluorescent label MDCC with the free thiol of Y446C or Q607C. (C) Coomassie stain and fluorescent scan of PBP2a-Y446C mutant before and after labeling with MDCC, followed by a desalting column to remove excess MDCC. Labeling of Q607C was confirmed in the same way. (D) Fluorescent emission scans of PBP2a-Y446C-MDCC or PBP2a-Q607C-MDCC in the absence (black) and in the presence of 20 µM ceftaroline after 5 (red), 10 (blue), and 15 (green) minutes.

Figure 3

Deconvoluted mass spectra from LC/MS analysis of the wild-type PBP2a in the presence of 100 µM imipenem (IPM) and 20 µM ceftaroline (CFT) (top) or no ceftaroline (CFT) (bottom). In the absence of ceftaroline, only the molecular ion for the apoenzyme is seen.