Daniel Lim

Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A

The facultative anaerobe Escherichia coli is able to assemble specific respiratory chains by synthesis of appropriate dehydrogenases and reductases in response to the availability of specific substrates. Under anaerobic conditions in the presence of nitrate, E. coli synthesizes the cytoplasmic membrane-bound quinol-nitrate oxidoreductase (nitrate reductase A; NarGHI), which reduces nitrate to nitrite and forms part of a redox loop generating a proton-motive force. We present here the crystal structure of NarGHI at a resolution of 1.9 Å. The NarGHI structure identifies the number, coordination scheme and environment of the redox-active prosthetic groups, a unique coordination of the molybdenum atom, the first structural evidence for the role of an open bicyclic form of the molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) cofactor in the catalytic mechanism and a novel fold of the membrane anchor subunit. Our findings provide fundamental molecular details for understanding the mechanism of proton-motive force generation by a redox loop.

Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus.

The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad-spectrum β-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Because of its low affinity for β-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at β-lactam concentrations that inhibit the β-lactam-sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 Å resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefin, penicillin G and methicillin allow, for the first time, a comparison of an apo and acylated resistant PBP. An analysis of the PBP2a active site in these forms reveals the structural basis of its resistance and identifies features in newly developed β-lactams that are likely important for high affinity binding.

Structural insight into the transglycosylation step of bacterial cell-wall biosynthesis.

Peptidoglycan glycosyltransferases (GTs) catalyze the polymerization step of cell-wall biosynthesis, are membrane-bound, and are highly conserved across all bacteria. Long considered the “holy grail” of antibiotic research, they represent an essential and easily accessible drug target for antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus. We have determined the 2.8 angstrom structure of a bifunctional cell-wall cross-linking enzyme, including its transpeptidase and GT domains, both unliganded and complexed with the substrate analog moenomycin. The peptidoglycan GTs adopt a fold distinct from those of other GT classes. The structures give insight into critical features of the catalytic mechanism and key interactions required for enzyme inhibition.

Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog

Sialic acid terminates oligosaccharide chains on mammalian and microbial cell surfaces, playing critical roles in recognition and adherence. The enzymes that transfer the sialic acid moiety from cytidine-5′-monophospho-N-acetyl-neuraminic acid (CMP-NeuAc) to the terminal positions of these key glycoconjugates are known as sialyltransferases. Despite their important biological roles, little is understood about the mechanism or molecular structure of these membrane-associated enzymes. We report the first structure of a sialyltransferase, that of CstII from Campylobacter jejuni, a highly prevalent foodborne pathogen. Our structural, mutagenesis and kinetic data provide support for a novel mode of substrate binding and glycosyl transfer mechanism, including essential roles of a histidine (general base) and two tyrosine residues (coordination of the phosphate leaving group). This work provides a framework for understanding the activity of several sialyltransferases, from bacterial to human, and for the structure-based design of specific inhibitors.

Structural and biochemical characterization of the type III secretion chaperones CesT and SigE.

Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir–CesT and enzymatic activity profiles of SigD–SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.

Crystal structure of the MexR repressor of the mexRAB-oprM multidrug efflux operon of Pseudomonas aeruginosa.

MexR is a member of the MarR family of bacterial transcriptional regulators and is the repressor for the MexAB-OprM operon, which encodes a tripartite multidrug efflux system inPseudomonas aeruginosa. Mutations in MexR result in increased resistance to multiple antibiotics due to overexpression of this efflux system. We have determined the crystal structure of MexR to 2.1-Å resolution in the absence of effector. The four copies of the MexR dimer in the asymmetric unit are observed in multiple conformations. Analysis of these conformational states in the context of a model of the MexR-DNA complex proposed in this study suggests that an effector-induced conformational change may inhibit DNA binding by reducing the spacing of the DNA binding domains. The inhibited conformation is exhibited by one of the four MexR dimers, which contains an ordered C-terminal tail from a neighboring monomer inserted between its DNA binding domains and which we propose may resemble the MexR-effector complex. Our results indicate that MexR may differ from the other described member of this family, MarR, in the nature of its effector, mode of DNA binding, and mechanism of regulation.

Crystal structure and kinetic analysis of beta-lactamase inhibitor protein-II in complex with TEM-1 beta-lactamase.

The structure of the 28 kDa β-lactamase inhibitor protein-II (BLIP-II) in complex with the TEM-1 β-lactamase has been determined to 2.3 Å resolution. BLIP-II is a secreted protein produced by the soil bacterium Streptomyces exfoliatus SMF19 and is able to bind and inhibit TEM-1 with subnanomolar affinity. BLIP-II is a seven-bladed β-propeller with a unique blade motif consisting of only three antiparallel β-strands. The overall fold is highly similar to the core structure of the human regulator of chromosome condensation (RCC1). Although BLIP-II does not share the same fold with BLIP, the first β-lactamase inhibitor protein for which structural data was available, a comparison of the two complexes reveals a number of similarities and provides further insights into key components of the TEM-1–BLIP and TEM-1–BLIP-II interfaces. Our preliminary results from gene knock-out studies and scanning electron microscopy also reveal a critical role of BLIP-II in sporulation.

Calpain silencing by a reversible intrinsic mechanism.

Uncontrolled activation of calpain can lead to necrotic cell death and irreversible tissue damage. We have discovered an intrinsic mechanism whereby the autolysis-generated protease core fragment of calpain is inactivated through the inherent instability of a key α-helix. This auto-inactivation state was captured by the 1.9 Å Ca2+-bound structure of the protease core from m-calpain, and sequence alignments suggest that it applies to about half of the calpain isoforms. Intact calpain large subunits are also subject to this inhibition, which can be prevented through assembly of the heterodimers. Other isoforms or their released cores are not silenced by this mechanism and might contribute to calpain patho-physiologies.

Structural analysis of an “open” form of PBP1B from Streptococcus pneumoniae

The class A PBP1b from Streptococcus pneumoniae is responsible for glycosyltransferase and transpeptidase (TP) reactions, forming the peptidoglycan of the bacterial cell wall. The enzyme has been produced in a stable, soluble form and undergoes time‐dependent proteolysis to leave an intact TP domain. Crystals of this TP domain were obtained, diffracting to 2.2 Å resolution, and the structure was solved by using molecular replacement. Analysis of the structure revealed an “open” active site, with important conformational differences to the previously determined “closed” apoenzyme. The active‐site nucleophile, Ser460, is in an orientation that allows for acylation by β‐lactams. Consistent with the productive conformation of the conserved active‐site catalytic residues, adjacent loops show only minor deviation from those of known acyl‐enzyme structures. These findings are discussed in the context of enzyme functionality and the possible conformational sampling of PBP1b between active and inactive states.

Heavy metal-mediated crystallization of Escherichia coli phytase and analysis of bridging interactions.

Escherichia coli phytase is a phosphatase that catalyzes the hydrolysis of phytic acid into inorganic phosphate and myo-inositol. Two crystal forms of this enzyme were obtained in the presence of heavy metals. Crystal forms I and II were obtained with the heavy atoms CdCl(2) and HgCl(2) and diffracted to 1.5 A and 2.25 A resolution, respectively. Hg(2+) and Cd(2+) both acted as molecular bridge(s), linking and stabilizing E. coli phytase in the unit cell, and played a crucial role in the crystallization of phytase by bridging neighbouring symmetry related molecules.