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.

Keywords: X-ray crystallography; allosteric mechanism; antibiotic resistance.

Fig. 1.

Domains of PBP2a and key ligands. (A) The chemical structures of a synthetic NAG-NAM(pentapeptide) (1) and ceftaroline (2). The R1 and R2 groups of 2 are labeled. (B) Ribbon representation of PBP2a acylated by ceftaroline. The N-terminal extension is colored in green, the remaining allosteric domain is colored in gold, and the transpeptidase (TP) domain is colored in blue. These domain colors are retained in all other figures. Two molecules of ceftaroline (capped sticks in red) are found in complex with protein: one covalently bound as an acyl-enzyme in the TP domain (CFT1) and one intact at the allosteric domain (CFT2). A muramic acid saccharide (capped sticks in magenta) is found at the center of the allosteric domain. The arrow indicates the point of attachment of the membrane anchor. (C) The solvent-accessible surface representation for PBP2a is shown. The distance between the two ceftaroline molecules is 60 Å. (D) Ribbon representation of PBP2a in complex with 1 (black sticks). This view is rotated ∼45° on the y axis compared with the view of C.

Fig. 2.

Interaction of PBP2a with ceftaroline at the active site. (A) View of ceftaroline within the active site is shown in capped sticks, with carbons in green, oxygens in red, nitrogens in blue, and sulfur in yellow. (B) Structural contrast between Complex 1 (pink tubes) and the PBP2a ceftaroline acyl-enzyme (blue tubes). Relevant amino acids are represented in capped sticks, and the two important active site loops are labeled. (C) Structural comparison of the ceftaroline interaction at the PBP2a active site compared with ceftobiprole. Superposition of the active site of ceftaroline-acyl-PBP2a (CFT-PBP2a) with ceftobiprole-acyl-PBP2a (PDB ID code 4DKI). Ceftaroline is drawn as green sticks, and ceftobiprole is drawn as red sticks. Side chains of residues are shown in capped sticks for the CFT-PBP2a (blue) and the ceftobiprole-acyl-PBP2a (brown). Polar interactions in ceftaroline-acyl-PBP2a are shown as dashed lines. *The disordered region found in the ceftobiprole-acyl-PBP2a around M641. The crystal structure of PBP2a in complex with ceftobiprole (PDB ID code 4DKI) also shows residual electron density at the same position in the allosteric domain as occupied by ceftaroline, and it also exhibits some of the modifications observed in the ceftaroline complex.

Fig. 3.

The structure of the allosteric domain and its interactions with ligands. (A) Interactions of the peptidoglycan 1 (capped sticks in magenta) bound at the allosteric site. (B) View of compound 1 (in magenta) bound at the allosteric site. We superimpose the apo PBP2a structure (PDB ID code 1VQQ) in gray onto this structure. The unique salt bridge interactions formed on 1 binding are represented as dotted lines. (C) View of ceftaroline (sticks with carbon atoms in dark red) bound noncovalently at the allosteric site. This complex also shows the muramic acid (MUR) saccharide (in cyan) at 1 o’clock. We superimpose the apo PBP2a structure in gray onto this structure. The unique salt bridge interactions formed on ceftaroline binding are represented as dotted lines. Mutations in clinical isolates for ceftaroline-resistant (N146 and E150) and ceftobiprole- or l-695,256–resistant (E150*, E239*, and E237*) are labeled. (D) Stereoview of the allosteric site. The structures of 1 (orange) and ceftaroline (red) are from our crystal structures. The computational model of the extended peptidoglycan is shown in green. (E) Composite model shown in D is based on the crystal coordinates for compound 1 (at 9 o’clock; blue) and the backbone atoms of ceftaroline (the boxed structure in red) for the d-Ala-d-Ala moiety at 3 o’clock (blue) per the hypothesis by Tipper and Strominger (21). The intervening atoms depicted in black correspond to the second NAG-NAM unit.

Fig. 4.

Stereoview of the allosteric signal propagation in PBP2a. Binding of the peptidoglycan (black) at the allosteric site propagates a unique network of salt bridge interactions extending between the allosteric and catalytic domains. The seven salt bridge interactions seen by crystallography are identified with arrowheads. An additional 17 salt bridge interactions were predicted by molecular dynamics (Table S2). The catalytic serine (yellow) and the acidic (red) and basic (blue) residues of the salt bridge interactions are shown as spheres. Peptidoglycan binding at the allosteric site stimulates this domino effect commencing from the allosteric site (Lobes 1 and 2) to Lobe 3 onto the β3–β4 loop. The changes in the β3–β4 loop of the active site as a result of formation of the unique salt bridge network are detailed in Fig. 2.