Mijoon Lee

How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function.

Significance Penicillin binding protein 2a imparts to the human pathogen Staphylococcus aureus resistance to β-lactam antibiotics. Our structural characterization of the allosteric basis governing its resistance mechanism identifies a basis for the design of new antibacterials that can both activate and inhibit this key resistance enzyme. 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.

Co-opting the Cell Wall in Fighting Methicillin-Resistant Staphylococcus aureus: Potent Inhibition of PBP 2a by Two Anti-MRSA β-Lactam Antibiotics

Methicillin-resistant Staphylococcus aureus (MRSA) is a global bacterial scourge that has become resistant to many classes of antibiotics, and treatment options for MRSA infections are limited. The cause of MRSA resistance to all commercially available beta-lactam antibiotics is the acquisition of the gene mecA, which encodes penicillin-binding protein 2a (PBP 2a). PBP 2a is a transpeptidase, which in contrast to the other transpeptidases of S. aureus does not experience inhibition by beta-lactam antibiotics. The lack of inhibition is due to a closed conformation for the active site for PBP 2a, which opens up only in the course of the catalytic function of the protein. Here we show that two new anti-MRSA antibiotics now undergoing clinical trials, ceftaroline and ME1036, are able to inhibit PBP 2a effectively, a process that is enhanced in the presence of a cell wall structural surrogate. It is likely that in the course of bacterial growth the occupancy of the allosteric site for the cell wall is co-opted by these antibiotics, and under these conditions the second-order rate constant for the encounter of the antibiotic and PBP 2a approaches the clinically useful value of 10(4)-10(5) M-1 s-1. These compounds are potent inhibitors of PBP 2a as well as PBPs from other species, and have potential as therapeutic agents for treatment of serious infections by MRSA and other resistant bacterial pathogens.

Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane

The outer membrane protein A (OmpA) plays important roles in anchoring of the outer membrane to the bacterial cell wall. The C‐terminal periplasmic domain of OmpA (OmpA‐like domain) associates with the peptidoglycan (PGN) layer noncovalently. However, there is a paucity of information on the structural aspects of the mechanism of PGN recognition by OmpA‐like domains. To elucidate this molecular recognition process, we solved the high‐resolution crystal structure of an OmpA‐like domain from Acinetobacter baumannii bound to diaminopimelate (DAP), a unique bacterial amino acid from the PGN. The structure clearly illustrates that two absolutely conserved Asp271 and Arg286 residues are the key to the binding to DAP of PGN. Identification of DAP as the central anchoring site of PGN to OmpA is further supported by isothermal titration calorimetry and a pulldown assay with PGN. An NMR‐based computational model for complexation between the PGN and OmpA emerged, and this model is validated by determining the crystal structure in complex with a synthetic PGN fragment. These structural data provide a detailed glimpse of how the anchoring of OmpA to the cell wall of gram‐negative bacteria takes place in a DAP‐dependent manner.—Park, J. S., Lee, W. C., Yeo, K. J., Ryu, K.‐S., Kumarasiri, M., Hesek, D., Lee, M., Mobashery, S., Song, J. H., Lim, S. I., Lee, J. C., Cheong, C., Jeon, Y. H., Kim, H.‐Y. Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram‐negative bacterial outer membrane. FASEB J. 26, 219–228 (2012). www.fasebj.org

Structural insights into the bactericidal mechanism of human peptidoglycan recognition proteins

Peptidoglycan recognition proteins (PGRPs) are highly conserved pattern-recognition molecules of the innate immune system that bind bacterial peptidoglycans (PGNs), which are polymers of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) cross-linked by short peptide stems. Human PRGPs are bactericidal against pathogenic and nonpathogenic Gram-positive bacteria, but not normal flora bacteria. Like certain glycopeptide antibiotics (e.g., vancomycin), PGRPs kill bacteria by directly interacting with their cell wall PGN, thereby interfering with PGN maturation. To better understand the bactericidal mechanism of PGRPs, we determined the crystal structure of the C-terminal PGN-binding domain of human PGRP-Iβ in complex with NAG-NAM-l-Ala-γ-d-Glu-l-Lys-d-Ala-d-Ala, a synthetic glycopeptide comprising a complete PGN repeat. This structure, in conjunction with the previously reported NMR structure of a dimeric PGN fragment, permitted identification of major conformational differences between free and PGRP-bound PGN with respect to the relative orientation of saccharide and peptide moieties. These differences provided structural insights into the bactericidal mechanism of human PGRPs. On the basis of molecular modeling, we propose that these proteins disrupt cell wall maturation not only by sterically encumbering access of biosynthetic enzymes to the nascent PGN chains, but also by locking PGN into a conformation that prevents formation of cross-links between peptide stems in the growing cell wall.

Recognition of peptidoglycan and β-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae.

The eukaryotic‐type serine/threonine kinase StkP from Streptococcus pneumoniae is an important signal‐transduction element that regulates the expression of numerous pneumococcal genes. We have expressed the extracellular C‐terminal domain of StkP kinase (C‐StkP), elaborated a three‐dimensional structural model and performed a spectroscopical characterization of its structure and stability. Biophysical experiments show that C‐StkP binds to synthetic samples of the cell wall peptidoglycan (PGN) and to β‐lactam antibiotics, which mimic the terminal portions of the PGN stem peptide. This is the first experimental report on the recognition of a minimal PGN unit by a PASTA‐containing kinase, suggesting that non‐crosslinked PGN may act as a signal for StkP function and pointing to this protein as an interesting target for β‐lactam antibiotics.

Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia

BackgroundCerebral ischemia has been shown to induce activation of matrix metalloproteinases (MMPs), particularly MMP-9, which is associated with impairment of the neurovasculature, resulting in blood–brain barrier breakdown, hemorrhage and neurodegeneration. We previously reported that the thiirane inhibitor SB-3CT, which is selective for gelatinases (MMP-2 and −9), could antagonize neuronal apoptosis after transient focal cerebral ischemia.ResultsHere, we used a fibrin-rich clot to occlude the middle cerebral artery (MCA) and assessed the effects of SB-3CT on the neurovasculature. Results show that neurobehavioral deficits and infarct volumes induced by embolic ischemia are comparable to those induced by the filament-occluded transient MCA model. Confocal microscopy indicated embolus-blocked brain microvasculature and neuronal cell death. Post-ischemic SB-3CT treatment attenuated infarct volume, ameliorated neurobehavioral outcomes, and antagonized the increases in levels of proform and activated MMP-9. Embolic ischemia caused degradation of the neurovascular matrix component laminin and tight-junction protein ZO-1, contraction of pericytes, and loss of lectin-positive brain microvessels. Despite the presence of the embolus, SB-3CT mitigated these outcomes and reduced hemorrhagic volumes. Interestingly, SB-3CT treatment for seven days protected against neuronal laminin degradation and protected neurons from ischemic cell death.ConclusionThese results demonstrate considerable promise for the thiirane class of selective gelatinase inhibitors as potential therapeutic agents in stroke therapy.

Elucidation of the Molecular Recognition of Bacterial Cell Wall by Modular Pneumococcal Phage Endolysin CPL-1 *□

Pneumococcal bacteriophage-encoded lysins are modular proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) in treatment of streptococcal infections. The first x-ray crystal structures of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1, in complex with three bacterial cell wall peptidoglycan (PG) analogues are reported herein. The Cpl-1 structure is folded in two well defined modules, one responsible for anchoring to the pneumococcal cell wall and the other, a catalytic module, that hydrolyzes the PG. Conformational rearrangement of Tyr-127 is a critical event in molecular recognition of a stretch of five saccharide rings of the polymeric peptidoglycan (cell wall). The PG is bound at a stretch of the surface that is defined as the peptidoglycan-binding sites 1 and 2, the juncture of which catalysis takes place. The peptidoglycan-binding site 1 binds to a stretch of three saccharides of the peptidoglycan in a conformation essentially identical to that of the peptidoglycan in solution. In contrast, binding of two peptidoglycan saccharides at the peptidoglycan-binding site 2 introduces a kink into the solution structure of the peptidoglycan, en route to catalytic turnover. These findings provide the first structural evidence on recognition of the peptidoglycan and shed light on the discrete events of cell wall degradation by Cpl-1.

Bacterial AmpD at the Crossroads of Peptidoglycan Recycling and Manifestation of Antibiotic Resistance

The bacterial enzyme AmpD is an early catalyst in commitment of cell wall metabolites to the recycling events within the cytoplasm. The key internalized metabolite of cell wall recycling, beta-D-N-acetylglucosamine-(1-->4)-1,6-anhydro-beta-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-DAP-D-Ala-D-Ala (compound 1), is a poor substrate for AmpD. Two additional metabolites, 1,6-anhydro-N-acetylmuramyl-peptidyl derivatives 2a and 2c, served as substrates for AmpD with a k(cat)/K(m) of >10(4) M(-1) s(-1). The enzyme hydrolytically processes the lactyl amide bond of the 1,6-anhydro-N-acetylmuramyl moiety. The syntheses of these substrates and other ligands are reported herein, which made the characterization of the enzymic reaction possible. Furthermore, it is documented that the enzyme is specific for both the atypical peptide stem of the cell wall fragments and the presence of the sterically encumbered 1,6-anhydro-N-acetylmuramyl moiety; hence it is a peptidase with a unique function in bacterial physiology. The implications of the function of this catalyst for the entry into the cell wall recycling events and the reversal of induction of the production of beta-lactamase, an antibiotic resistance determinant, are discussed.

Reactions of all Escherichia coli lytic transglycosylases with bacterial cell wall.

The reactions of all seven Escherichia coli lytic transglycosylases with purified bacterial sacculus are characterized in a quantitative manner. These reactions, which initiate recycling of the bacterial cell wall, exhibit significant redundancy in the activities of these enzymes along with some complementarity. These discoveries underscore the importance of the functions of these enzymes for recycling of the cell wall.

Synthetic peptidoglycan motifs for germination of bacterial spores.

Certain Gram-positive bacteria—as exemplified by Bacillus anthracis, the causative agent of anthrax—can produce dormant and environmentally resistant spores under conditions of nutritional limitation. These spores exit from dormancy via the process of germination that is triggered by exposure to specific molecules.[1] While the precise chemical nature of these molecules, known as germinants, varies according to the organism, they are typically nutrients.[2] Recently, we reported that supernatants from cultures of growing bacteria and constituents of the cell wall could serve as germinants of dormant B. subtilis and B. anthracis spores.[3] Since fragments of the cell wall are released in the course of bacterial growth, the presence of these molecules in the milieu as germination signals might be physiologically relevant.