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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Curr Med Chem. 2015;22(14):1678–1686. doi: 10.2174/0929867322666150311150215

The Allosteric Site for the Nascent Cell Wall in Penicillin-Binding Protein 2a: an Achilles’ Heel of Methicillin-Resistant Staphylococcus aureus

Ivan Acebrón 1, Mayland Chang 2, Shahriar Mobashery 2, Juan A Hermoso 1,*
PMCID: PMC4686279  NIHMSID: NIHMS744567  PMID: 25760091

Abstract

The ability to resist the effect of a wide range of antibiotics makes methicillin-resistant Staphylococcus aureus (MRSA) a leading global human pathogen. A key determinant of resistance to β-lactam antibiotics in this organism is penicillin-binding protein 2a (PBP2a), an enzyme that catalyzes the crosslinking reaction between two adjacent peptide stems during the peptidoglycan biosynthesis. The recently published crystal structure of the complex of PBP2a with ceftaroline, a cephalosporin antibiotic that shows efficacy against MRSA, has revealed the allosteric site at 60-Å distance from the transpeptidase domain. Binding of ceftaroline to the allosteric site of PBP2a triggers conformational changes that lead to the opening of the active site from a closed conformation, where a second molecule of ceftaroline binds to give inhibition of the enzyme. The discovery of allostery in MRSA remains the only known example of such regulation of cell-wall biosynthesis and represents a new paradigm in fighting MRSA. This review summarizes the present knowledge of the allosteric mechanism, the conformational changes allowing PBP2a catalysis and the means by which some clinical strains have acquired resistance to ceftaroline by disrupting the allosteric mechanism.

Keywords: Methicillin-resistant Staphylococcus aureus, antibiotic resistance, penicillin-binding proteins, allosteric mechanism, β-lactam antibiotics, conformational change, X-ray crystallography

INTRODUCTION

Infectious diseases are a leading cause of deaths worldwide. The increasing incidence and widespread emergence of pathogens that are resistant to antibiotics have reversed advances in the treatment of many infections [1]. The return to the pre-antibiotic era is a current reality. Without effective antibiotics the practice of modern medicine as we know it will cease [2]. This problem is especially important in management of infections arising after chemotherapy, surgery and organ transplantations, among other procedures [3]. Nowadays infections by resistant bacteria result not only in higher rates of death, but patients who survive stay in the hospital longer, have prolonged recuperation and long-term disability [3]. In the European Union, antibacterial resistance causes 25,000 deaths per year, with estimated costs of €1,500 million per year [2]. In the United States, at least 23,000 deaths occur every year due to antibiotic-resistant infections [3]. Tackling the problem of antibiotic resistance requires understanding of the mechanisms that bacteria have evolved to survive in the presence of antibiotics. Basic research in antibiotic resistance will open doors to new pharmacological approaches to combat resistant pathogens [4].

One of the most serious pathogens is bacterium Staphylococcus aureus, which currently represents a major problem in both the clinical and community settings. Staphylococcus aureus is a Gram-positive bacterium, responsible for skin and respiratory tract infections. Variants of this organism exist that are resistant to a wide range of β-lactam antibiotics, known as methicillin-resistant S. aureus or MRSA [5]. MRSA infections are difficult to treat, with a mortality rate of ~20%, and are the leading cause of death by a single infectious agent in the USA, accounting for more deaths than the HIV [6].

PBP2A: A KEY DETERMINANT IN RESISTANCE TO β-LACTAM ANTIBIOTIC IN STAPHYLOCOCCUS AUREUS

During the 1940s, the β-lactam antibiotic penicillin was extensively used to treat S. aureus infections. Soon after introduction of penicillin to the clinic, resistance appeared and disseminated rapidly. As of the 1960s approximately 80% of isolated strains in Western countries exhibited resistance to the antibiotic due to the endogenous production of the S. aureus β-lactamase PC1. The second generation of penicillins, which included methicillin and oxacillin, among others, was introduced in the late 1950s and was intended to be stable toward the β-lactamase activity. However, shortly after their introduction to the clinic, a strain of S. aureus was identified in the United Kingdom with resistance to methicillin [7]. This organism came to be known as MRSA. Within a couple of years, MRSA was distributed globally and remains a problem to the present day. Not only S. aureus had become resistance to methicillin, but also, as it turned out, to the entire class of penicillins and cephalosporins that were known at the time, and to the carbapenems, which were discovered later. Although newer classes of antibiotics (vancomycin [8], daptomycin [9], ceftaroline [10] and oxazolidinones [11]) have been developed for treatment of MRSA, and notwithstanding the strict controls that are implemented for their use, resistance to all has emerged [12].

The bacterial cell wall is unique to the bacterium, providing structural integrity and protection from lysis due to high intracellular osmotic pressure. The enzymes involved in cell-wall biosynthesis are the targets of β-lactams antibiotics [13]. The cell wall is primarily comprised of peptidoglycan, which consists of repeating linear polymers of β-linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), with stem peptides that are attached to the NAM unit. The peptide stems (l-Ala-γ-d-Glu-l-Lys(Gly)5-d-Ala-d-Ala in S. aureus; with the pentaglycyl moiety appended to the side chain of l-Lys) of neighboring peptidoglycan strands are the points of crosslinking. One of the most important proteins involved in peptidoglycan synthesis is the family of penicillin-binding proteins (PBPs). As such, PBPs have emerged as important targets in antibiotic development.

PBPs are membrane-bound and perform peptidoglycan transpeptidation (crosslinking), carboxypeptidation (for regulation of the degree of crosslinking and other reasons) and transglycosylation (in the case of bifunctional enzymes) (Fig. (1)). PBPs have been classified traditionally in two different main groups: the high-molecular-mass (HMM) PBPs and the low-molecular-mass (LMM) PBPs (Fig. (2)). There are two types of HMM PBPs depending on their catalytic activity and their modular structure, designated as classes A and B [14]. Class A PBPs are able to catalyze both the polymerization of sugar chains (transglycosylation) and crosslinking (transpeptidation) reactions due to the presence of a glycosyltransfer domain (GT), followed by a transpeptidase domain (TP). Class B enzymes are, however, monofunctional with only the transpeptidase activity. Within these two classes there are several groups that have different subdomain arrangements that allow classification of PBPs as shown in Fig (2). Apart from the HMM PBPs, there exist also LMM PBPs, also referred to as class C PBPs. These are single-domain proteins involved in a variety of biological processes, such as cell division, peptidoglycan remodeling and recycling [15].

Fig. (1).

Fig. (1)

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Schematic representation of the two main reactions catalyzed by HMM PBPs during the peptidoglycan synthesis: transpeptidation and transglycosylation. As a result, PBPs assemble the peptidoglycan on the surface of the cytoplasmic membrane in two sequential steps.

Fig. (2).

Fig. (2)

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Classification of PBPs by molecular mass and by domain composition, according to Macheboeuf et al. (2006) [16]. PBP2a belongs to the last group of class B PBPs. Proteins are anchored to the membrane through a transmembrane helix (TM) or an amphipatic helix. Depending on the PBP, different domains such as N-terminal domain (N-term), PASTA domains, Nuclear transport Factor 2-like domain (NTF2) or C-terminal domain (C-term) can be identified.

Tipper and Strominger argued that β-lactam antibiotics inhibit the dd-transpeptidase activity of PBPs by mimicking the acyl-d-Ala-d-Ala terminus of the peptidoglycan stem peptide [17]. This mimicry, leads to covalent and essentially irreversible modification of the active-site serine of the transpeptidase domain [17]. From the point of view of the kinetic mechanism (Fig. (3)), antibiotics exhibit good acylation rate constants (k2 values), while the corresponding deacylation rate constants (k3 values [18]) tend to be very small and, hence, the reaction produces an irreversible covalent complex (Fig. (3)). In the particular case of MRSA, β-lactam resistance arises due to the insensitivity of PBP2a (encoded by the mecA gene) to this inhibition [19], allowing cell-wall biosynthesis and survival in the presence of antibiotics [20].

Fig. (3).

Fig. (3)

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Reaction scheme for hydrolysis of β-lactam antibiotics showing the minimalist kinetic mechanism. In general, β-lactams react irreversibly with the transpeptidase domain of the PBPs, producing a covalent intermediate (acyl-PBP) that inhibits the enzyme.

In PBP2a, the covalent modification of the catalytic serine (S403) by these antibiotics does not occur as readily as in other PBPs, because the inaccessibility to the active site, which is reflected in poor acylation rate constants (t1/2 = 3–12 min) [21, 22]. As disclosed by Villegas-Estrada et al., the kinetic parameters of PBP2a inhibition change in the presence of certain ligands such as synthetic peptidoglycan fragments and some antibiotics. The higher the concentration of these ligands, the more attenuated the dissociation constant (Kd) for the β-lactam binding, suggesting that the active site of PBP2a may became more available for antibiotic acylation [21]. This effect is also supported by circular dichroism measurements, which document a conformational change in protein in the presence of these ligands. These observations were interpreted in light of allosteric involvement by this enzyme that enables catalysis in cell-wall biosynthesis [21]. Yet, the closed conformation of the active site also explains resistance to β-lactam antibiotics, which cannot gain access to the active site. As such, allostery facilitates access to the active site for the physiological substrates, while keeping out the antibiotic.

STRUCTURAL INFORMATION ON PBP2A

The crystal structure of PBP2a [23] without the transmembrane anchor (residues 1–23), exhibits a multidomain arrangement (Fig (4)) with the catalytic domain (residues 327–668) that shares the overall fold with other transpeptidases [15]. The transpeptidase domain is made up of two different subdomains, a five stranded β-sheet capped by three α-helices and an all-helical fold. The active site is situated at the interface of these two subdomains. Similar to other PBPs, the catalytic residue S403 is placed in a narrow cleft at the N-terminus of helix α2 in the characteristic sequence motif S-X-X-K and is proximal to the strand β3. In the ligand-free state, S403 is well sheltered within the active site in a tight groove and is also hydrogen bonded with K406, which enhances the nucleophilicity of the Oγ of S403 for catalysis [23, 24].

Fig. (4).

Fig. (4)

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Crystallographic three-dimensional structure of PBP2a (PDB 1VQQ). Each subdomain is depicted in different colors indicating their sequence extension: N-terminal extension (green), allosteric domain (orange) and transpeptidase domain (blue). These domain colors are retained in all other figures. The lobes comprising the allosteric site are labeled, as is the active site.

Comparison of the unliganded PBP2a (PDB 1VQQ) with diverse antibiotic-complexed crystal structures solved up to date [23, 25, 26] has revealed that PBP2a undergoes a slight conformational change at strand β3 and at helix α2 of the N-terminus for acylation to take place. Practically, the enzyme would have to accommodate the two strands of the peptidoglycan within the active site for the crosslinking reaction to proceed. This encounter requires a volume in excess of 1,000 Å3 within the active-site cavity, which is considerably larger than that needed for antibiotic binding. This has not been observed in any of these crystallographic structures.

THE UNPRECEDENTED ALLOSTERIC MECHANISM THAT ENABLES PHYSIOLOGICAL FUNCTION

In contrast to the majority of β-lactam antibiotics tested so far, PBP2a is readily inhibited by ceftaroline (Fig. (5)), the most recent fifth-generation cephalosporin approved by the FDA [27]. Crystal structures of PBP2a in the presence of ceftaroline (PDB codes 3ZFZ and 3ZG0) and of a muropeptide (PDB code 3ZG5) [25] (Fig. (6)), as well as kinetic measurements [22] provided insights to the mode of action of this antibiotic. The crystal structures of PBP2a-ceftaroline complexes revealed an active site where the catalytic serine S403 is acylated by one ceftaroline molecule (CFT1 in Fig. (6B, 6D)) [25]. The electron density found inside the allosteric site at the intersection of Lobe 1 (166–240), Lobe 2 (258–277), Lobe 3 (364–390) and the top of the N-terminal extension domain (27–138), allowed modeling a non-covalently bound second ceftaroline molecule (CFT2 in Fig. (6B, 6E)).

Fig. (5).

Fig. (5)

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Chemical structures of the novel β-lactam antibiotics ceftaroline and ceftobiprole. The R1 and R2 groups are depicted in green and purple, respectively.

Fig. (6).

Fig. (6)

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Active and allosteric sites in PBP2a. Ribbon representation of PBP2a in complex with (A) muropeptide (MP) and (B) ceftaroline (CFT). (C) Close-up view of the MP-binding at the allosteric domain. (D and E) Ceftaroline-binding at the active site and the allosteric site, respectively. The complex shown in (E) also has a molecule of muramic acid (Mur) saccharide (in deep purple) at 1 o’clock. Mutations in clinical isolates for ceftaroline-resistance are labeled with an asterisk (*).

Despite the significant separation of the allosteric site and the transpeptidase active site (~60 Å), the allosteric site is able to transmit a signal to the catalytic domain that alters the protein conformation and leads to opening of the cavity at the active site. The crystal structure of PBP2a complexed with a muropeptide and ceftaroline shows a salt-bridge network connecting the allosteric site to the catalytic domain (Fig (7)). The concerted motion of these residues transmits the signal from the allosteric to the catalytic site in a process akin to falling dominos. The hallmark of allostery is protein conformational flexibility. This is normally reflected in numerous conformers interchangeable at different timescales that involve amino-acid networks connecting distant sites [28]. In PBP2a, this flexibility is observed in the different lobes that make up the allosteric domain. Notwithstanding, the conformational changes originate in the allosteric site since the initial position of the lobes experiences a displacement. For instance, as shown in the complex with a synthetic fragment of the peptidoglycan (Fig (6)) [25], the distance between the lobes shortens by 3 Å by increasing the number of salt-bridge interactions. Interestingly, the protein reorganization depends on the effector bound to the allosteric site, since the observed conformational change is different between the complexes with the synthetic peptidoglycan (PDB 3ZG5, Fig. (6A, 6C)) and the antibiotic ceftaroline.

Fig. (7).

Fig. (7)

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Number of salt bridges connecting the allosteric site with the active site in PBP2a structures. (A) New salt bridges connecting the allosteric site with the active site in the PBP2a:peptidoglycan complex as calculated by molecular-dynamics simulations [25]. Basic residues are represented as blue spheres and acidic residues as red spheres. (B) The number of salt bridges connecting Lobe 1 with Lobe 2 (yellow), Lobe 1 with Lobe 3 (light orange) and Lobe 3 with β3–β4 loop (dark orange), increases from the PBP2a:muropeptide complex to the PBP2a:ceftaroline complex. As predicted by molecular simulations, these interactions are largely increased when PBP2a interacts with the peptidoglycan at the allosteric site.

The conformational reorganization that happens within the active site upon the allosteric effector’s binding involves numerous residues located in the α2-α3 and β3–β4 loops. As a result of these structural changes, the active-site volume increases from 007E;500 Å3 to 1,300 Å3. The displacement of the β3–β4 loop increases the length of the active site from 10 Å to 23 Å, which would allow acylation and accommodation of the peptidoglycan peptide stems. This new conformation allows S403 to act as a nucleophile and initiates catalysis, as S403 becomes more exposed and better positioned to react.

In addition to ceftaroline, ceftobiprole (Fig. (5)) also possesses anti-MRSA activity. While the crystal structure of PBP2a acylated by ceftobiprole [26] shows similar structural changes in the β3 strand and the N-terminus of the α2 helix to those observed in the PBP2a-ceftaroline complex, the E602-R612 salt bridge that is made as a result of interaction with the R1 group of ceftaroline and the recognition of the R2 group of both antibiotics is different.

THE PHYSIOLOGICAL ROLE OF THE ALLOSTERIC SIT

A computational model based on the X-ray structures of the muropeptide and ceftaroline bound to the allosteric site and of the solution NMR structure of the peptidoglycan was constructed [25]. This complex for binding of the nascent peptidoglycan (the substrate for the catalytic domain) accommodates a hexasaccharide backbone, which identifies two distinct stem-peptide-binding surfaces (Fig. (8)). The glycan chain of the peptidoglycan is also perfectly accommodated by Lobes 1 and 2 (Fig. (8)), therefore indicating that ceftaroline at the allosteric site is somehow mimicking the peptide stem of peptidoglycan, once it is bound to this site [25], consistent with the Tipper-Strominger hypothesis [17]. In this context, the nascent peptidoglycan with the full-length stem peptide could work as the natural allosteric effector triggering opening of the active site of the transpeptidase domain through communications between Lobes 1 and 2, with Lobe 3 and with the β3–β4 loop of the active site (Fig. (8)). Indeed, molecular-dynamics simulations indicated that greater the peptidoglycan chain bound at the allosteric site would result in both larger displacements in the lobes of the allosteric domain and more salt bridges connecting Lobes 1 and 2 with the β3–β4 loop (Fig (7)). These computational results are supportive of the previous kinetic studies that observed an elevation of the acylation rate constant in the presence of synthetic peptidoglycan fragments [21].

Fig. (8).

Fig. (8)

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The allosteric site in PBP2a could recognize the nascent peptidoglycan (PG) chains. Computational model of a PG chain (glycan chains as blue sticks and peptide-stems as pink sticks) on PBP2a (molecular surface). The second peptide-stem of PG directly superimposes with ceftaroline at the allosteric site (CFT2). Ceftaroline at the active site (CFT1) is labeled, as well as the different PBP2a regions.

S. aureus possesses a total of four PBPs (PBP1, PBP2, PBP3 and PBP4). The MRSA variants also have PBP2a (Fig. (9)). The crystallographic structures of all of them are already known [23, 29–31], with the exception of PBP1, which is a close homologue of the protein PBP2× from Streptococcus pneumoniae [32]. Proteins PBP1–3 act as transpeptidases even though PBP2 is the only bifunctional enzyme identified in S. aureus. Deletion mutations have demonstrated that PBP3 is not critical for bacterial survival since PBP1 and PBP2 may functionally replace its function [33]. As for PBP4, it is the only LMM PBP in S. aureus, while the rest belong to the HMM group. It is noteworthy that none of the solved X-ray structures for the other PBPs of S. aureus to date have even a hint of the allosteric domain.

Fig. (9).

Fig. (9)

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Penicillin-binding proteins from S. aureus. Structural comparison of PBPs solved from S. aureus and one PBP from Streptococcus pneumoniae. S. aureus has four PBPs (PBP1, PBP2, PBP3 and PBP4) and one extra in the MRSA strain (PBP2a). PBP1 has been replaced by S. pneumoniae PBP2×, a structural homologue. The transpeptidase domain is shown in blue, while the other domains are shown in orange.

DISRUPTION OF THE ALLOSTERIC COMMUNICATION AS AN ANTIBIOTIC-RESISTANCE MECHANISM

Considering the long distance between the allosteric and active sites, disruption of communication between the two could disrupt the processes of catalysis by PBP2a. Thus, 14 mutant variants of PBP2a in three different regions along the identified path of the conformational change triggered by ceftaroline were designed (Fig (10)) [25]. Most of the mutants exhibited diminished capacity to undergo the conformational change that is the requisite to acylation within the active site and two mutants (K387A-D635 and D343A-E389A-D635A) showed no acylation activity even though the overall protein fold was conserved (Fig (10)). These results support the thesis for the need for conformational change in regions of the protein remote from the active site to support catalysis. Moreover, we also observed that effects are larger as mutations are closer to the active site. For instance, natural occurring point mutations Y446N and E447K lying just within the binding site of the catalytic domain lead to dramatic results in normal ceftaroline activation [34]. We have asserted that there might be multiple routes for the propagation of the allosteric trigger with some redundancy to accommodate the convergence of the orchestral motion that leads to the opening of the active site.

Fig. (10).

Fig. (10)

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The PBP2a mutants altering charges along the activation path. Effects on acylation at the active site are indicated by the color code. Mutated residues are represented as spheres (right) and are labeled.

Despite the short time that ceftaroline has been in the clinic, MRSA has already found a way to resist the antibiotic by altering the allosteric communication. Recently, two clinical variants of PBP2a were isolated from MRSA strains that have shown ceftaroline resistance. The variants include a double mutant (N146K-E150K) and a triple mutant (N146K-E150K-H351N) that have two mutations in common that are distant from the active site. Analyses of the sequences have revealed that both point mutations (N146K and E150K) are located in the allosteric site, previously identified by Otero et al. [25]. In fact, the crystal structure of the clinical double mutant [35] shows a completely different network of intramolecular interactions that involves residues from the three lobes whose intersection comprises the allosteric domain. These mutations change the nearby environment of the unliganded PBP2a, interfering with the propagation of the signal from the allosteric site to the active site. The outcome is resistance to ceftaroline by an unprecedented mechanism, namely interference with the fidelity of the allosteric response.

As indicated, the propagation of the allosteric signal depends on the reorganization of the ion-paired side chains of a series of acidic and basic residues. If the initial orientation of these residues has changed, it might affect the subsequent conformational change upon ceftaroline binding at the allosteric site. The mutated residues K146 and K150 create new salt bridges and turn the negative electrostatic potential on the molecular surface to a more basic one (Fig (11)). This would affect the initial recognition of the allosteric effector avoiding the conformational change that opens the active-site cavity. The alteration of the interaction network for the allosteric effect occurs by creating new salt bridges that are not present in the wild-type PBP2a. This altered connectivity within the allosteric site seen in the X-ray structures is consistent with the effects of the kinetic measurements [35]. Therefore, the clinical double mutations at positions 146 and 150 not only alter the pattern of interactions around the mutated positions, but also tamper with the salt-bridge network among many other residues within Lobe-2 and Lobe-3, as far away as 35 Å from the mutated residues.

Fig. (11).

Fig. (11)

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Comparison of the electrostatic surfaces of the wild-type PBP2a (A) and the clinical mutant N146K/E150K double mutant (B). Acidic regions are colored in red and basic in blue. Mutations in the clinical isolate are labeled as well as Lobe-1 and Lobe-2. As a result of both mutations, the electrostatic potential changes drastically. The Mur molecule found at the allosteric site in the double mutant is depicted in yellow-capped sticks. The electrostatic potential has been calculated by the APBS program [36] and the PBP2PQR server [37].

CONCLUSION

Staphylococcus aureus has developed a grand repertoire of mechanisms to neutralize antibiotics. The first identified mechanism was the enzymatic hydrolysis of β-lactams by the production of PC1 β-lactamase, but subsequently many other mechanisms have been reported involving a diverse number of gene products directly related with antibiotics resistance. In addition to β-lactams, S. aureus exhibits resistance to glycopeptides, quinolones and aminoglycosides among other antimicrobial agents [38] each of these resistance mechanisms presenting specific features Shahriar, Mayland algun review que podamos citar?. One of the most recent mechanisms, found in MRSA, was the incorporation of the gen mecA from another undetermined bacteria to provide β-lactam resistance. The gene product is a unique PBP (PBP2a) capable to be regulated by allostery.

Allostery is increasingly being identified in disparate regulatory processes, such as regulation of proteins (e.g., hemoglobin [39], glycogen phosphorylase [40]), transport across the membrane (e.g., nAChR [41]), signal transmission (e.g., G-protein-coupled receptors [42]) and intercellular communications [41]. Allosteric sites are also used in the design of novel drugs for different diseases, such as cancer (e.g., the use of taxol as an antitumor agent [43]), Alzheimer’s disease (e.g., inhibition of GSK-3β by using allosteric modulators such as thiadiazolindindione (TDZD) derivates [44]), hyperglycemia (e.g., activation of human liver pyruvate kinase [45]), etc. These drugs are usually designed to stabilize one of the conformations so as to avoid the allosteric transition and therefore clog or hyper-activate the biological function. Regarding cell-wall biosynthesis, allostery has not been investigated in MRSA so far but the unique function of PBP2a provides opportunities to do it now. The discovery of the nature of this particular allosteric mechanism identifies it as an Achilles’ heel for this pathogen. Disruption of allostery has the potential for novel antibiotic design in the future.

ACKNOWLEDGMENTS

This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2011-25326) to J.A.H., the biomedicine program of government of autonomous community of Madrid (S2010/BMD-2457) to J.A.H., the National Institutes of Health (AI090818 to M.C. and S.M., and AI104987 to S.M.)

ABBREVIATIONS

HMM

High Molecular Mass

IC50

Half maximal inhibitory concentration

k1, k2 & k3

First order rate constants for PBP acylation-deacylation mechanism

Kd

Dissociation constant

MIC

Minimum Inhibitory Concentration

MRSA

Methicillin-Resistant Staphylococcus aureus

LMM

Low Molecular Mass

PBP

Penicillin-Binding Protein

Footnotes

CONFLICT OF INTEREST

The author(s) confirm that this article content has no conflicts of interest.

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