Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the d-Ala-d-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. β-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a β-lactamase and is not trapped as an acyl intermediate with β-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.Penicillin binding proteins (PBPs) are critical components of the cell wall synthesis machinery in bacteria. These membrane-associated proteins are broadly classified as low-molecular-mass (LMM) PBPs that are monofunctional d,d-carboxypeptidase enzymes or multimodular high-molecular-mass (HMM) PBPs with multiple functional roles. PBPs, in general, are anchored to the cytoplasmic membrane by a noncleavable pseudo-signal peptide. In the case of the HMM PBPs, the cytoplasmic C-terminal domain binds penicillin and catalyzes peptidoglycan cross-linking, whereas the juxtamembrane N-terminal domain participates in transglycosylation (12). The catalytic penicillin-binding (PB) module also occurs as part of penicillin sensor transducers, such as Staphylococcus aureus MecR and Bacillus licheniformis BlaR (15). The transpeptidase activity in HMM PBPs is based on a conserved lysine residue located in the so-called catalytic S-X-X-K motif, whereas the other conserved S-X-N and K(H)-T(S)-G motifs govern carboxypeptidase activity and bind penicillin (20). The carboxypeptidase domain of PBPs is the target for β-lactam antibiotics in susceptible staphylococci (with penicillin MICs as low as 1 μg/ml).The transpeptidase activity of the PBPs occurs at the d-Ala-d-Ala terminus of a precursor disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-l-Ala-d-Ala-l-Lys-d-Ala-d-Ala. This reaction is initiated by acylation involving a nucleophilic attack by the active-site serine on the penultimate d-Ala residue to form an acyl-enzyme complex. The C-terminal d-Ala is subsequently released from the peptide chain, followed by deacylation. In the case of HMM PBPs, deacylation occurs when an amino group on a second peptide substrate acts as an acceptor, resulting in a peptide cross-link between two adjacent peptidoglycan strands. The carboxypeptidase activity of LMM PBPs follows a similar reaction scheme, except that the acceptor in this case is a water molecule. β-Lactam antibiotics mimic the substrates of the PBPs. However, unlike the natural substrate, the β-lactam-PBP acyl adduct is stable and results in irreversible inhibition of PBP function. The β-lactam-PBP acyl adduct has been characterized extensively, with over 50 protein-antibiotic complexes reported to date (37). Thus, in contrast to the nonessential LMM PBPs, HMM PBPs constitute lethal targets for β-lactam antibiotics (6).Staphylococcus aureus is a gram-positive coccus and is one of the leading causes of high morbidity and mortality associated with both community- and hospital-associated infections (42, 46). This coccus shows extensive genomic variation, with over 22% of the genome dedicated to dispensable regions. A genome-scale analysis of a clinical strain of S. aureus is of particular interest in this context, wherein the conversion of a susceptible strain of S. aureus to a multidrug-resistant phenotype was shown to involve just 35 mutations in 13 loci, achieved within 3 months (36). Of the five PBPs in S. aureus, an acquired PBP, PBP2a, is the most extensively examined, as it was noted to be a specific marker for methicillin-resistant S. aureus (MRSA) strains. Among the intrinsic PBPs, PBP1 has been shown to play a key role in cell growth and division (2). PBP2 is a dual-function enzyme with both transglycosylase and transpeptidase activities, and inhibition of this protein leads to restrained peptidoglycan elongation and subsequent leakage of cytoplasmic contents due to cell lysis (34, 40). Inactivation of PBP3 neither changes the muropeptide composition of the cell wall nor significantly decreases the rate of autolysis. However, cells of abnormal size and shape and with disoriented septa are produced when bacteria with inactivated PBP3 are grown with sub-MIC levels of methicillin (29).S. aureus PBP4 is a carboxypeptidase and is needed for the secondary cross-linking of peptidoglycan (19). However, it is not essential for cell growth under laboratory conditions, because mutants of S. aureus defective in PBP4 are viable (48). Overexpression of PBP4 was noted to result in an increase in β-lactam resistance and in greater cross-linking of the peptidoglycan (18). S. aureus PBP4 is similar to other LMM PBPs and is grouped within the superfamily of penicillin-susceptible and penicillin-interacting enzymes. However, homologues of PBP4 have a different phenotype in other species (1, 15). For example, a mutation of PBP4 in Pseudomonas aeruginosa triggers an AmpR-dependent overproduction of the chromosomal β-lactamase AmpC. The P. aeruginosa PBP4 mutant also activates CreBC, a two-component regulator, thereby mediating β-lactam resistance (33). Indeed, S. aureus PBP4 has been suggested to have different functions in strains with different genetic backgrounds (26). However, based on in vitro and genetic data, S. aureus PBP4 is primarily a transpeptidase and has little d,d-carboxypeptidase activity. This is also supported by the observation that increased carboxypeptidase activity decreases cell wall cross-linking due to loss of the free d-Ala-d-Ala termini necessary for transpeptidation (10). In this context, it is pertinent that pbp4 gene knockout strains of S. aureus were more resistant to the glycopeptide antibiotic vancomycin (46).Here we present the biochemical and structural characteristics of PBP4 from S. aureus strain COL. S. aureus PBP4 is a β-lactamase. A comparison of the crystal structure of S. aureus PBP4 in complex with antibiotic with that of its Escherichia coli homologue, PBP5, provides a conformational and biochemical rationale for the β-lactamase activity of PBP4. Monitoring the expression of PBP4 in the MRSA strain COL and representative clinical strains of S. aureus suggested that the expression level of PBP4 does not fluctuate substantially across these strains. Together, these data on the structure, expression, activity, and regulation of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.