Disruption of allosteric response as an unprecedented mechanism of resistance to antibiotics

Abstract

Ceftaroline, a recently approved β-lactam antibiotic for treatment of infections by methicillin-resistant Staphylococcus aureus (MRSA), is able to inhibit penicillin-binding protein 2a (PBP2a) by triggering an allosteric conformational change that leads to the opening of the active site. The opened active site is now vulnerable to inhibition by a second molecule of ceftaroline, an event that impairs cell-wall biosynthesis and leads to bacterial death. The triggering of the allosteric effect takes place by binding of the first antibiotic molecule 60 Å away from the active site of PBP2a within the core of the allosteric site. We document, by kinetic studies and by determination of three X-ray structures of the mutant variants of PBP2a that result in resistance to ceftaroline, that the effect of these clinical mutants is the disruption of the allosteric trigger in this important protein in MRSA. This is an unprecedented mechanism for antibiotic resistance.

Figure 1

Chemical structures of ceftaroline, nitrocefin, ceftobiprole, and L-695,256.

Figure 2

(A) Molecular surface of PBP2a. The N-terminal extension is colored in green, the remaining allosteric domain in yellow and the transpeptidase domain in blue. Active site is indicated by an arrow at 1 o’clock. The locations of the point mutations are indicated in magenta and are labeled. Lobes 1 (L-1), 2 (L-2), and 3 (L-3) of the allosteric site are labeled. The reported allosteric ligands, ceftaroline (CFT) and synthetic peptidoglycan (C1; not discussed in the manuscript) (PDB ID codes 3ZG0 and 3ZG5, respectively) are superimposed and depicted as black-capped sticks. (B) The backbone of the N146K/E150K double mutant (orange) is superimposed onto that of the wild-type PBP2a structure (PDB ID code 1VQQ colored in gray). The muramic acid (MUR) found at the allosteric site in the N146K/E150K mutant is depicted in green-capped sticks for the carbon atoms. Upon mutation, the backbone and the network of salt-bridge interactions are altered. Residues involved in these changes in L-2 are represented in capped sticks (in orange for the mutant and in gray for the wild-type PBP2a). New salt-bridges and hydrogen bonds are shown as dashed lines.

Figure 3

Comparison between the electrostatic potential on the molecular surface of (A) wild-type PBP2a and (B) the clinical N146K/E150K double mutant. Acidic regions are colored in red and basic in blue. The locations of the point mutations are labeled. Lobes 1 (L-1) and 2 (L-2) are labeled. The mutations at positions 146 and 150 provoke a strong change in the electrostatic potential in the entire allosteric site. The new allosteric site presenting a marked basic character extending from the intact ceftaroline binding-site (see Figure 2A) to the MUR binding-site. The muramic acid (MUR) found at the allosteric site in the N146K/E150K mutant is depicted in yellow-capped sticks for the carbon atoms.