I03-Macromolecular Crystallography
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Open Access
Abstract: Microbes that have evolved to live on lignocellulosic biomass face unique challenges in the effective and efficient use of this material as food. The bacterium Shewanella sp. ANA-3 has the potential to utilize arabinan and arabinoxylan, and uptake of the monosaccharide, l-arabinose, derived from these polymers, is known to be mediated by a single ABC transporter. We demonstrate that the substrate binding protein of this system, GafASw, binds specifically to l-arabinofuranose, which is the rare furanose form of l-arabinose found in lignocellulosic biomass. The structure of GafASw was resolved to 1.7 Å and comparison to Escherichia coli YtfQ (GafAEc) revealed binding site adaptations that confer specificity for furanose over pyranose forms of monosaccharides, while selecting arabinose over another related monosaccharide, galactose. The discovery of a bacterium with a natural predilection for a sugar found abundantly in certain lignocellulosic materials suggests an intimate connection in the enzymatic release and uptake of the sugar, perhaps to prevent other microbes scavenging this nutrient before it mutarotates to l-arabinopyranose. This biological discovery also provides a clear route to engineer more efficient utilization of plant biomass components in industrial biotechnology.
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Mar 2023
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: Peptide transporters play important nutritional and cell signalling roles in Bacillus subtilis, which are pronounced during stationary phase adaptations and development. Three high-affinity ATP-binding cassette (ABC) family transporters are involved in peptide uptake – the oligopeptide permease (Opp), another peptide permease (App) and a less well-characterized dipeptide permease (Dpp). Here we report crystal structures of the extracellular substrate binding proteins, OppA and DppE, which serve the Opp and Dpp systems, respectively. The structure of OppA was determined in complex with endogenous peptides, modelled as Ser-Asn-Ser-Ser, and with the sporulation-promoting peptide Ser-Arg-Asn-Val-Thr, which bind with Kd values of 0.4 and 2 µM, respectively, as measured by isothermal titration calorimetry. Differential scanning fluorescence experiments with a wider panel of ligands showed that OppA has highest affinity for tetra- and penta-peptides. The structure of DppE revealed the unexpected presence of a murein tripeptide (MTP) ligand, l-Ala-d-Glu-meso-DAP, in the peptide binding groove. The mode of MTP binding in DppE is different to that observed in the murein peptide binding protein, MppA, from Escherichia coli, suggesting independent evolution of these proteins from an OppA-like precursor. The presence of MTP in DppE points to a role for Dpp in the uptake and recycling of cell wall peptides, a conclusion that is supported by analysis of the genomic context of dpp, which revealed adjacent genes encoding enzymes involved in muropeptide catabolism in a gene organization that is widely conserved in Firmicutes.
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Dec 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.
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Oct 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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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.
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Jul 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349–712 has been crystallized and its structure has been determined to reveal a core eight-stranded β-barrel fold with a neighbouring subdomain containing a five-stranded β-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission.
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Jun 2020
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Andrew S.
Bell
,
Zhiyong
Yu
,
Jennie A.
Hutton
,
Megan H.
Wright
,
James A.
Brannigan
,
Daniel
Paape
,
Shirley M.
Roberts
,
Charlotte L.
Sutherell
,
Markus
Ritzefeld
,
Anthony
Wilkinson
,
Deborah F
Smith
,
Robin
Leatherbarrow
,
Edward W.
Tate
Diamond Proposal Number(s):
[1221, 7864]
Abstract: The leishmaniases, caused by Leishmania species of protozoan parasites, are neglected tropical diseases with 12-15 million cases worldwide. Current therapeutic approaches are limited by toxicity, resistance and cost. N-Myristoyltransferase (NMT), an enzyme ubiquitous and essential in all eukaryotes, has been validated via genetic and pharmacological methods as a promising antileishmanial target. Here we describe a comprehensive structure activity relationship study of a thienopyrimidine series previously identified in a high throughput screen against Leishmania NMT, across 68 compounds in enzyme- and cell-based assay formats. Using a chemical tagging target engagement biomarker assay we identify the first inhibitor in this series with on-target NMT activity in leishmania parasites. Furthermore, crystal structure analyses of 12 derivatives in complex with Leishmania major NMT revealed key factors important for future structure-guided optimization delivering IMP-105 (43), a compound with modest activity against L. donovani intracellular amastigotes and excellent selectivity (>660-fold) for Leishmania NMT over human NMTs.
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Jun 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Abstract: Peptides play an important signalling role in Bacillus subtilis, where their uptake by one of two ABC-type oligopeptide transporters, Opp and App, is required for efficient sporulation. Homologues of these transporters in Clostridium difficile have been characterized, but their role, and hence that of peptides, in regulating sporulation in this organism is less clear. Here, the oligopeptide-binding receptor proteins for these transporters, CdAppA and CdOppA, have been purified and partially characterized, and the crystal structure of CdAppA has been determined in an open unliganded form. Peptide binding to either protein could not be observed in Thermofluor assays with a set of ten peptides of varying lengths and compositions. Re-examination of the protein sequences together with structure comparisons prompts the proposal that CdAppA is not a versatile peptide-binding protein but instead may bind a restricted set of peptides. Meanwhile, CdOppA is likely to be the receptor protein for a nickel-uptake system.
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Apr 2019
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I02-Macromolecular Crystallography
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Nadine
Daou
,
Yuanguo
Wang
,
Vladimir M.
Levdikov
,
Madhumitha
Nandakumar
,
Jonathan
Livny
,
Laurent
Bouillaut
,
Elena
Blagova
,
Keshan
Zhang
,
Boris R.
Belitsky
,
Kyu
Rhee
,
Anthony J.
Wilkinson
,
Xingmin
Sun
,
Abraham L.
Sonenshein
Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.
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Jan 2019
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[9333]
Abstract: The influence of statistics on calculated lattice strains has been studied by comparing crystal plasticity finite element (CPFE) calculations with strains measured experimentally. Experimentally, when Bragg's law is obeyed, a plane normal must lie within a narrow orientation range (∼ 0.02° for synchrotron diffraction), or Bragg tolerance. However, CPFE models consider only a small number of grains compared to experiments, necessitating a justification of the statistically representative volume. It also becomes necessary to assess the threshold of Bragg tolerance allowable for the determined statistically representative volume. In this study, an 8 × 8 × 8 model was deemed as statistically representative such that only small benefits are obtained in terms of lattice strain calculations by adopting larger models such as 10 × 10 × 10. Based on the selected model, an allowable Bragg tolerance of approximately 5° was calculated. Also highlighted was the coupling between lattice strain, texture, hardening and applied boundary condition which are discriminators that will affect the choice of model size and Bragg tolerance threshold.
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May 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Aurélie
Mousnier
,
Andrew S.
Bell
,
Dawid P.
Swieboda
,
Julia
Morales-Sanfrutos
,
Inmaculada
Perez-Dorado
,
James A.
Brannigan
,
Joseph
Newman
,
Markus
Ritzefeld
,
Jennie A.
Hutton
,
Anabel
Guedán
,
Amin S.
Asfor
,
Sean W.
Robinson
,
Iva
Hopkins-Navratilova
,
Anthony J.
Wilkinson
,
Sebastian L.
Johnston
,
Robin J.
Leatherbarrow
,
Tobias J.
Tuthill
,
Roberto
Solari
,
Edward W.
Tate
Diamond Proposal Number(s):
[12579, 7864, 9948]
Abstract: Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.
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May 2018
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