I02-Macromolecular Crystallography
|
Abstract: The murein peptide amidase MpaA is a cytoplasmic enzyme that processes peptides derived from the turnover of murein. We have purified the enzyme from Escherichia coli and demonstrated that it efficiently hydrolyses the ?-D-glutamyl-diaminopimelic acid bond in the murein tripeptide (L-Ala-?-D-Glu-meso-Dap), with Km and kcat values of 0.41±0.05 mM and 38.3±10 s?1. However, it is unable to act on the murein tetrapeptide (L-Ala-?-D-Glu-meso-Dap-D-Ala). E. coli MpaA is a homodimer containing one bound zinc ion per chain, as judged by mass spectrometric analysis and size-exclusion chromatography. To investigate the structure of MpaA we solved the crystal structure of the orthologous protein from Vibrio harveyi to 2.17 Å (1Å=0.1 nm). Vh_MpaA, which has identical enzymatic and biophysical properties to the E. coli enzyme, has high structural similarity to eukaryotic zinc carboxypeptidases. The structure confirms that MpaA is a dimeric zinc metalloprotein. Comparison of the structure of MpaA with those of other carboxypeptidases reveals additional structure that partially occludes the substrate-binding groove, perhaps explaining the narrower substrate specificity of the enzyme compared with other zinc carboxypeptidases. In ?-proteobacteria mpaA is often located adjacent to mppA which encodes a periplasmic transporter protein previously shown to bind murein tripeptide. We demonstrate that MppA can also bind murein tetrapeptide with high affinity. The genetic coupling of these genes and their related biochemical functions suggest that MpaA amidase and MppA transporter form part of a catabolic pathway for utilization of murein-derived peptides that operates in ?-proteobacteria in addition to the established murein recycling pathways.
|
Dec 2012
|
|
I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[306, 7864]
Abstract: Solvent organization is a key but underexploited contributor to the thermodynamics of protein–ligand recognition, with implications for ligand discovery, drug resistance and protein engineering. Here, we explore the contribution of solvent to ligand binding in the Haemophilus influenzae virulence protein SiaP. By introducing a single mutation without direct ligand contacts, we observed a >1000-fold change in sialic acid binding affinity. Crystallographic and calorimetric data of wild-type and mutant SiaP showed that this change results from an enthalpically unfavourable perturbation of the solvent network. This disruption is reflected by changes in the normalized atomic displacement parameters of crystallographic water molecules. In SiaP’s enclosed cavity, relative differences in water-network dynamics serve as a simple predictor of changes in the free energy of binding upon changing protein, ligand or both. This suggests that solvent structure is an evolutionary con-straint on protein sequence that contributes to ligand affinity and selectivity.
|
Sep 2019
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18598, 13587]
Open Access
Abstract: Body odour is a characteristic trait of Homo sapiens, however its role in human behaviour and evolution is poorly understood. Remarkably, body odour is linked to the presence of a few species of commensal microbes. Herein we discover a bacterial enzyme, limited to odour-forming staphylococci that are able to cleave odourless precursors of thioalcohols, the most pungent components of body odour. We demonstrated using phylogenetics, biochemistry and structural biology that this cysteine-thiol lyase (C-T lyase) is a PLP-dependent enzyme that moved horizontally into a unique monophyletic group of odour-forming staphylococci about 60 million years ago, and has subsequently tailored its enzymatic function to human-derived thioalcohol precursors. Significantly, transfer of this enzyme alone to non-odour producing staphylococci confers odour production, demonstrating that this C-T lyase is both necessary and sufficient for thioalcohol formation. The structure of the C-T lyase compared to that of other related enzymes reveals how the adaptation to thioalcohol precursors has evolved through changes in the binding site to create a constrained hydrophobic pocket that is selective for branched aliphatic thioalcohol ligands. The ancestral acquisition of this enzyme, and the subsequent evolution of the specificity for thioalcohol precursors implies that body odour production in humans is an ancient process.
|
Jul 2020
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
|
Andrew
Bell
,
Jason
Brunt
,
Emmanuelle
Crost
,
Laura
Vaux
,
Ridvan
Nepravishta
,
C. David
Owen
,
Dimitrios
Latousakis
,
An
Xiao
,
Wanqing
Li
,
Xi
Chen
,
Martin A.
Walsh
,
Jan
Claesen
,
Jesus
Angulo
,
Gavin H.
Thomas
,
Nathalie
Juge
Abstract: Sialic acid (N-acetylneuraminic acid (Neu5Ac)) is commonly found in the terminal location of colonic mucin glycans where it is a much-coveted nutrient for gut bacteria, including Ruminococcus gnavus. R. gnavus is part of the healthy gut microbiota in humans, but it is disproportionately represented in diseases. There is therefore a need to understand the molecular mechanisms that underpin the adaptation of R. gnavus to the gut. Previous in vitro research has demonstrated that the mucin-glycan-foraging strategy of R. gnavus is strain dependent and is associated with the expression of an intramolecular trans-sialidase, which releases 2,7-anhydro-Neu5Ac, rather than Neu5Ac, from mucins. Here, we unravelled the metabolism pathway of 2,7-anhydro-Neu5Ac in R. gnavus that is underpinned by the exquisite specificity of the sialic transporter for 2,7-anhydro-Neu5Ac and by the action of an oxidoreductase that converts 2,7-anhydro-Neu5Ac into Neu5Ac, which then becomes a substrate of a Neu5Ac-specific aldolase. Having generated an R. gnavus nan-cluster deletion mutant that lost the ability to grow on sialylated substrates, we showed that—in gnotobiotic mice colonized with R. gnavus wild-type (WT) and mutant strains—the fitness of the nan mutant was significantly impaired, with a reduced ability to colonize the mucus layer. Overall, we revealed a unique sialic acid pathway in bacteria that has important implications for the spatial adaptation of mucin-foraging gut symbionts in health and disease.
|
Oct 2019
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Membrane bound acyltransferase-3 (AT3) domain-containing proteins are implicated in a wide range of carbohydrate O-acyl modifications, but their mechanism of action is largely unknown. O-antigen acetylation by AT3 domain-containing acetyltransferases of Salmonella spp. can generate a specific immune response upon infection and can influence bacteriophage interactions. This study integrates in situ and in vitro functional analyses of two of these proteins, OafA and OafB (formerly F2GtrC), which display an “AT3-SGNH fused” domain architecture, where an integral membrane AT3 domain is fused to an extracytoplasmic SGNH domain. An in silico-inspired mutagenesis approach of the AT3 domain identified seven residues which are fundamental for the mechanism of action of OafA, with a particularly conserved motif in TMH1 indicating a potential acyl donor interaction site. Genetic and in vitro evidence demonstrate that the SGNH domain is both necessary and sufficient for lipopolysaccharide acetylation. The structure of the periplasmic SGNH domain of OafB identified features not previously reported for SGNH proteins. In particular, the periplasmic portion of the interdomain linking region is structured. Significantly, this region constrains acceptor substrate specificity, apparently by limiting access to the active site. Coevolution analysis of the two domains suggests possible interdomain interactions. Combining these data, we propose a refined model of the AT3-SGNH proteins, with structurally constrained orientations of the two domains. These findings enhance our understanding of how cells can transfer acyl groups from the cytoplasm to specific extracellular carbohydrates.
|
Aug 2020
|
|
I04-Macromolecular Crystallography
|
Open Access
Abstract: Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates.
|
Nov 2015
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Abstract: The human gut symbiont Ruminococcus gnavus scavenges host‐derived N‐acetylneuraminic acid (Neu5Ac) from mucins, by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D nuclear magnetic resonance (NMR), we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-DANA intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram negative and Gram positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionisation spray mass spectrometry (ESI-MS) that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolise 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry (DSF) analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli was dependent on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.
|
Jul 2020
|
|
I22-Small angle scattering & Diffraction
|
Diamond Proposal Number(s):
[14792]
Open Access
Abstract: We report the thermodynamically controlled growth of solution-processable and free-standing nanosheets via peptide assembly in two dimensions. By taking advantage of self-sorting between peptide β-strands and hydrocarbon chains, we have demonstrated the formation of Janus 2D structures with single-layer thickness, which enable a predetermined surface heterofunctionalization. A controlled 2D-to-1D morphological transition was achieved by subtly adjusting the intermolecular forces. These nanosheets provide an ideal substrate for the engineering of guest components (e.g., proteins and nanoparticles), where enhanced enzyme activity was observed. We anticipate that sequence-specific programmed peptides will offer promise as design elements for 2D assemblies with face-selective functionalization.
|
Sep 2017
|
|
I24-Microfocus Macromolecular Crystallography
|
Open Access
Abstract: Mammals produce volatile odours that convey different types of societal information. In Homo sapiens, this is now recognised as body odour, a key chemical component of which is the sulphurous thioalcohol, 3-methyl-3-sulfanylhexan-1-ol (3M3SH). Volatile 3M3SH is produced in the underarm as a result of specific microbial activity, which act on the odourless dipeptide-containing malodour precursor molecule, S-Cys-Gly-3M3SH, secreted in the axilla (underarm) during colonisation. The mechanism by which these bacteria recognise S-Cys-Gly-3M3SH and produce body odour is still poorly understood. Here we report the structural and biochemical basis of bacterial transport of S-Cys-Gly-3M3SH by Staphylococcus hominis, which is converted to the sulphurous thioalcohol component 3M3SH in the bacterial cytoplasm, before being released into the environment. Knowledge of the molecular basis of precursor transport, essential for body odour formation, provides a novel opportunity to design specific inhibitors of malodour production in humans.
|
Jul 2018
|
|
