Journal of Molecular Biology
Volume 396, Issue 3, 26 February 2010, Pages 634-645
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Crystal Structures of Penicillin-Binding Proteins 4 and 5 from Haemophilus influenzae

https://doi.org/10.1016/j.jmb.2009.11.055Get rights and content

Abstract

We have determined high-resolution apo crystal structures of two low molecular weight penicillin-binding proteins (PBPs), PBP4 and PBP5, from Haemophilus influenzae, one of the most frequently found pathogens in the upper respiratory tract of children. Novel β-lactams with notable antimicrobial activity have been designed, and crystal structures of PBP4 complexed with ampicillin and two of the novel molecules have also been determined. Comparing the apo form with those of the complexes, we find that the drugs disturb the PBP4 structure and weaken X-ray diffraction, to very different extents. PBP4 has recently been shown to act as a sensor of the presence of penicillins in Pseudomonas aeruginosa, and our models offer a clue to the structural basis for this effect. Covalently attached penicillins press against a phenylalanine residue near the active site and disturb the deacylation step. The ready inhibition of PBP4 by β-lactams compared to PBP5 also appears to be related to the weaker interactions holding key residues in a catalytically competent position.

Introduction

Peptidoglycan (also known as murein) forms an essential layer within the bacterial cell wall, both controlling the shape of the cell and protecting it from osmotic shock.1, 2, 3 During bacterial growth, peptidoglycan is constantly remodeled and new material is synthesized at specific locations.4, 5, 6, 7 The building block of peptidoglycan, called lipid II, is composed of an undecaprenyl-linked disaccharide–pentapeptide containing β-linked N-acetylglucosamine and N-acetylmuramic acid. The N-acetylmuramic acid residue is attached to the five-residue stem peptide. After translocation to the cytoplasmic side of the inner membrane, the disaccharide–pentapeptide undergoes transglycosylation8, 9 and transpeptidation (TP)10, 11 reactions, adding new material to the peptidoglycan layer by extending glycan chains cross-linked by the peptides. These essential reactions are carried out by penicillin-binding proteins (PBPs). TP occurs by the enzyme recognizing the C-terminal d-alanyl-d-alanine group on the peptide, forming an acyl–enzyme intermediate at the active serine and releasing d-alanine. A second peptidoglycan stem peptide attached to a neighboring glycan chain is then attached via its third amino residue, releasing the free enzyme. Peptidoglycan maturation occurs through release of the fifth C-terminal d-alanine residue of some peptide chains by dd-carboxypeptidases, which therefore prevents cross-linking. Endopeptidases are responsible for breaking peptide cross-links to allow the insertion of new peptidoglycan chains as well as recycling of the peptidoglycan. A comprehensive review of the known structures of PBPs, their classification, and role in peptidoglycan synthesis has recently been published by Sauvage et al.12

β-Lactam antibiotics are suicide substrates for PBPs whose four-membered ring moiety mimics the d-alanyl-d-alanine moiety of the stem peptide.13 Nucleophilic attack on β-lactams by the active-site serine residue of a PBP leads to ring opening, leaving the antibiotic covalently attached to the serine, blocking further transpeptidase activity and hence leading to cell death. PBPs are divided into two classes: high molecular weight (HMW) and low molecular weight (LMW) PBPs.12 The HMW PBPs can be divided into two subclasses based on sequence; HMW class A PBPs are bifunctional enzymes with transglycosylase and transpeptidase activity, whereas HMW class B PBPs have an N-terminal domain of unknown function and a C-terminal transpeptidase domain. Since the genes encoding the PBPs of H. influenzae were first cloned, at least seven have been identified: 1a (gene name: mrcA), 1b (mrcB), 2 (mrdA), 3 (ftsI), 4 (dacB), 5 (dacA), and 7 (pbpG). PBP1a and PBP1b are class A HMW PBPs, and PBP2 and PBP3 are class B. PBPs 4, 5, and 7 belong to the LMW PBPs, and comparison with the Escherichia coli homologues suggests that PBP5 has dd-carboxypeptidase activity and PBP7 has dd-endopeptidase activity. LMW PBP4 from E. coli (EcPBP4) is bifunctional in vivo14 and in vitro,15 and its single active site carries out both reactions.15 Sequence conservation of PBP4 between the two species strongly suggests that the proteins have similar enzyme activities. The LMW PBPs are in general not essential for bacterial survival but control the morphology of the cell and some play a role in cell division.3, 16, 17, 18 Although their role is more poorly understood than that of the HMW PBPs, the LMW PBPs can play significant roles in pathogenesis.19, 20

The transpeptidase, dd-endopeptidase, and dd-carboxypeptidase activities of all PBPs, regardless of class, depend upon three conserved sequence motifs, SxxK, SxN, and KTG, clustered around the active site. All crystal structures of PBPs show similar active sites, though their substrate preferences may vary. In the case of Actinomadura R39 dd-peptidase, for example, a surface pocket has been shown to bind the third residue of the stem peptide.21 Mutagenesis of the equivalent site in EcPBP4 has a substantial effect on both its enzyme activities.15 PBP3 of Neisseria gonorrhoeae, however, shows very high kcat and high KM with peptidoglycan mimetic peptide substrates, implying that despite the rapid turnover of this enzyme, there is only weak recognition of substrate features distant from the bond hydrolyzed.22 Beyond the initial acylation step, there is little evidence of a universal mechanism among PBPs. The active-site serine, in the SxxK motif, is acylated and deacylated on each turnover of the enzyme. TP occurs when the acyl intermediate undergoes nucleophilic attack by a peptide amine group, whereas in endopeptidases and dd-carboxypeptidases, attack is by water. The mechanism has been best studied in R61 from Streptomyces23, 24, 25 and PBP5 from E. coli (EcPBP5),26, 27, 28, 29 but the deacylation step, with either peptidoglycan substrates or β-lactam inhibitors (and the factors controlling its rate), is poorly understood, and recent attempts to mimic the evolution of a β-lactamase from a PBP have served to highlight how much is left to learn about the mechanism of these enzymes.30 A further complication is the possibility that deacylation may proceed by a different mechanism with β-lactam antibiotics than with natural substrates. Over three decades ago, Frere et al. showed that Streptomyces R61 differs from other PBPs in that it can cause fragmentation of the penicillin nucleus.31

Widespread misuse of antibiotics has caused increasing prevalence of antibiotic-resistant bacteria, which are often associated with life-threatening disease, but β-lactams remain important treatments for infection. Resistance may arise from either modified PBPs themselves or the expression of lactamases, which have evolved to hydrolyze β-lactams. However, PBPs are attractive and validated targets for antibiotic design since they are both essential and unique to bacteria. They are also located outside the cytoplasmic membrane and readily accessible by small-molecule inhibitors. Despite the huge effort directed toward understanding these proteins over several decades, new aspects of their biology continue to appear. It was recently revealed, for example, that PBP4 (dacB) of Pseudomonas aeruginosa functions as a trigger of β-lactamase expression when inhibited, through an unknown mechanism presumably involving the detection of an altered pattern of recycled peptidoglycan fragments.32 Insight into the structural basis of the sensitivity of different PBPs to inhibition has greatly improved recently but also remains imperfectly understood,33 as does the apparent lack of substrate specificity among the endopeptidases.21 EcPBP4 is known to be unusually sensitive to inhibition by β-lactams. To help find a structural mechanism for the sensitivity of PBP4, and hopefully to assist the development of new antibiotics, we have determined the X-ray crystal structures of PBPs 4 and 5 from Haemophilus influenzae and tested new β-lactams against this organism.

Section snippets

Results

Fifty novel β-lactams were designed by selecting different cyclic side groups to attach to the β-lactam ring. These side groups were selected for ease of synthesis and to give final compounds with a molecular mass in the range 300–500 Da. To examine the interactions made by these compounds and PBP4, we crystallized the protein in the apo form and we soaked crystals with the antibiotic prior to X-ray data collection. Apo-PBP4 crystals grew in space group P21 with a dimer in the asymmetric unit

Discussion

β-Lactams remain a very important group of antibiotics despite the prevalence of resistance among gram-positive and gram-negative bacteria. Among gram negatives, the principal means of resistance is the expression of lactamases such as AmpC, a chromosomally encoded enzyme that efficiently hydrolyzes β-lactams. AmpC expression is strongly induced by some of these molecules, such as cefetoxin or imipenem, but much more weakly by others such as piperacillin or cephalosporins. Until recently, LMW

HiPBP4

The region of the dacB gene encoding the globular portion of H. influenzae PBP4 (minus the N-terminal, hydrophobic, 27-amino-acid residue signal peptide) was amplified by PCR from genomic DNA. The PCR product was digested with NdeI and XhoI, respectively, and ligated into similarly cut pET28 vector with no histidine tag (Novagen). The pET28_HiPBP4 plasmid was transformed into E. coli BL21(DE3) star/pLysS. Bacteria were grown in LB medium containing 50 mg ml 1 kanamycin and 35 mg ml 1

Acknowledgements

We thank staff at beamlines BL-5A and BL-17A at Photon Factory for assistance with data collection. S.-Y.P. is supported in part by the ISS Applied Research Partnership Program, Maura Foods & Biosciences Inc., and Confocal Science Inc. This work was supported in part by a Medical Research Council collaboration grant to D.I.R. and grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan to J.R.H.T.

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