I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Haiyang
Wu
,
Emmanuelle H.
Crost
,
C. David
Owen
,
Wouter
Van Bakel
,
Ana
Martínez Gascueña
,
Dimitrios
Latousakis
,
Thomas
Hicks
,
Samuel
Walpole
,
Paulina A.
Urbanowicz
,
Didier
Ndeh
,
Serena
Monaco
,
Laura
Sánchez Salom
,
Ryan
Griffiths
,
Raven S.
Reynolds
,
Anna
Colvile
,
Daniel I. R.
Spencer
,
Martin
Walsh
,
Jesus
Angulo
,
Nathalie
Juge
Open Access
Abstract: The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-β-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.
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Dec 2021
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I03-Macromolecular Crystallography
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Christopher M.
Furze
,
Ignacio
Delso
,
Enriqueta
Casal
,
Collette S.
Guy
,
Chloe
Seddon
,
Chelsea M.
Brown
,
Hadyn L.
Parker
,
Anjana
Radhakrishnan
,
Raul
Pacheco-Gomez
,
Phillip J.
Stansfeld
,
Jesus
Angulo
,
Alexander D.
Cameron
,
Elizabeth
Fullam
Diamond Proposal Number(s):
[19880]
Open Access
Abstract: The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodelling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, STD NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays and the synthesis of trehalose analogues. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogues, and specifies a framework that can be exploited for the design of new anti-tubercular agents and/or diagnostic tools.
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Jan 2021
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
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.
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Jul 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Haiyang
Wu
,
Osmond
Rebello
,
Emmanuelle H.
Crost
,
C. David
Owen
,
Samuel
Walpole
,
Chloe
Bennati-Granier
,
Didier
Ndeh
,
Serena
Monaco
,
Thomas
Hicks
,
Anna
Colvile
,
Paulina A.
Urbanowicz
,
Martin A.
Walsh
,
Jesus
Angulo
,
Daniel I. R.
Spencer
,
Nathalie
Juge
Open Access
Abstract: The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1–3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3′-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.
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Apr 2020
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[17580]
Abstract: Surface hydrophobization of cellulose nanomaterials has been used in the development of nanofiller reinforced polymer composites and formulations based on Pickering emulsions. Despite well known effect of hydrophobic domains on self assembly or association of water soluble polymer amphiphiles, very few studies have addressed the behavior of hydrophobized cellulose nanomaterials in aqueous media. In this study, we investigate the properties of hydrophobized cellulose nanocrystals (CNCs) and their self assembly and amphiphilic properties in suspensions and gels. CNCs of different hydrophobicity were synthesized from sulfated CNCs by coupling primary alkylamines of different alkyl chain lengths (6, 8 and 12 carbon atoms). The synthetic route permitted the retention of surface charge, ensuring good colloidal stability of hydrohobized CNCs in aqueous suspensions. We compare surface properties (surface charge, Zeta potential), hydrophobicity (water contact angle, microenvironment probing using pyrene fluorescence emission) and surface activity (tensiometry) of different hydrophobized CNCs and hydrophilic CNCs. Association of hydrophobized CNCs driven by hydrophobic effects is confirmed by X ray scattering (SAXS) and autofluorescent spectroscopy experiments. As a result of CNC association, CNCs suspensions/gels can be produced with a wide range of rheological properties depending on the hydrophobic/hydrophilic balance. In particular, sol gel transitions for hydrophobized CNCs occur at lower concentrations then hydrophilic CNCs and more robust gels are formed by hydrophobized CNCs. Our work illustrates that amphiphilic CNCs can complement associative polymers as modifiers of rheological properties of water based systems.
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Jan 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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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.
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Oct 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The Mtb pathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. Mtb UgpB (Rv2833c) is the substrate binding domain of the UgpABCE transporter that recognizes glycerophosphocholine (GPC), indicating that this transporter has a role in recycling glycerophospholipid metabolites. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography, we report the structural analysis of Mtb UgpB complexed with GPC and have identified that Mtb UgpB not only recognizes GPC but is also promiscuous for a broad range of glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis precisely define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the Mtb UgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC, therefore optimizing the ability of Mtb to scavenge scarce nutrients and essential glycerophospholipid metabolites via a single transporter during intracellular infection.
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Aug 2019
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[163641]
Open Access
Abstract: A novel mechanism of heat-triggered gelation for oxidised cellulose nanofibrils (OCNF) is reported. We demonstrate that a synergistic approach combining rheology, small-angle X-ray scattering (SAXS) and saturation transfer difference NMR (STD NMR) experiments enables a detailed characterisation of gelation at different length scales. OCNF dispersions experience an increase in solid-like behaviour upon heating as evidenced by rheological studies, associated with enhanced interfibrillar interactions measured using SAXS. Interactions result in an increased fibrillar overlap and increased population of confined water molecules monitored by STD NMR. By comparison, cationic cellulose nanofibrils (produced by reaction of cellulose with trimethylglycidylammonium chloride) were found to be heat-unresponsive.
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Sep 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Abstract: Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1‐phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 using natural acceptor glucose and non‐cognate donor substrate ‐mannose 1‐phosphate (Man1P). To investigate how the enzyme recognizes different sugar 1‐phosphates, we solved the X‐ray crystal structures of PsLBP in complex with Glc1P and Man1P, providing the first molecular detail of the recognition of a non‐cognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxyl groups at C2, C4 and C6 of sugar 1‐phosphates. Furthermore, we used STD NMR to support the crystallographic studies on the sugar 1‐phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and the disaccharide products.
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Jun 2018
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I03-Macromolecular Crystallography
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Saannya
Sequeira
,
Devon
Kavanaugh
,
Donald A.
Mackenzie
,
Tanja
Šuligoj
,
Samuel
Walpole
,
Charlotte
Leclaire
,
A. Patrick
Gunning
,
Dimitrios
Latousakis
,
William G. T.
Willats
,
Jesus
Angulo
,
Changjiang
Dong
,
Nathalie
Juge
Diamond Proposal Number(s):
[9475]
Open Access
Abstract: Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host–microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.
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Mar 2018
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