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12 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural and functional characterisation of a stable, broad-specificity multimeric sialidase from the oral pathogen Tannerella forsythia Marianne J. Satur , Paulina A. Urbanowicz , Daniel I. R. Spencer , John Rafferty , Graham P. Stafford Biochemical Journal 479, 1785 - 1806 DOI: 10.1042/BCJ20220244 Diamond Proposal Number(s): [17773, 12788] Open Access Show Abstract ▼ Abstract: Sialidases are glycosyl hydrolase enzymes targeting the glycosidic bond between terminal sialic acids and underlying sugars. The NanH sialidase of Tannerella forsythia, one of the bacteria associated with severe periodontal disease plays a role in virulence. Here, we show that this broad-specificity enzyme (but higher affinity for α2,3 over α2,6 linked sialic acids) digests complex glycans but not those containing Neu5,9Ac. Furthermore, we show it to be a highly stable dimeric enzyme and present a thorough structural analysis of the native enzyme in its apo-form and in complex with a sialic acid analogue/ inhibitor (Oseltamivir). We also use non-catalytic (D237A) variant to characterise molecular interactions while in complex with the natural substrates 3- and 6-siallylactose. This dataset also reveals the NanH carbohydrate-binding module (CBM, CAZy CBM 93) has a novel fold made of antiparallel beta-strands. The catalytic domain structure contains novel features that include a non-prolyl cis-peptide and an uncommon arginine sidechain rotamer (R306) proximal to the active site. Via a mutagenesis programme, we identified key active site residues (D237, R212 and Y518) and probed the effects of mutation of residues in proximity to the glycosidic linkage within 2,3 and 2,6-linked substrates. These data revealed that mutagenesis of R306 and residues S235 and V236 adjacent to the acid–base catalyst D237 influence the linkage specificity preference of this bacterial sialidase, opening up possibilities for enzyme engineering for glycotechology applications and providing key structural information that for in silico design of specific inhibitors of this enzyme for the treatment of periodontitis.	Sep 2022
I04-Macromolecular Crystallography	The stringent response inhibits 70S ribosome formation in staphylococcus aureus by impeding GTPase-ribosome interactions Daniel J. Bennison , Jose A. Nakamoto , Timothy D. Craggs , Pohl Milón , John B. Rafferty , Rebecca M. Corrigan Mbio DOI: 10.1128/mBio.02679-21 Diamond Proposal Number(s): [17773] Open Access Show Abstract ▼ Abstract: During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. We show that RA-GTPases preferentially bind to 5′-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both in vitro and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control.	Nov 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Characterisation of a tripartite α-pore forming toxin from Serratia marcescens Alicia M. Churchill-Angus , Thomas Schofield , Thomas R. Marlow , Svetlana E. Sedelnikova , Jason S. Wilson , John B. Rafferty , Patrick J. Baker Scientific Reports 11 DOI: 10.1038/s41598-021-85726-0 Diamond Proposal Number(s): [17773, 24447] Open Access Show Abstract ▼ Abstract: Tripartite members of the ClyA family of α-PFTs have recently been identified in a number of pathogenic Gram-negative bacteria, including the human pathogen Serratia marcescens. Structures of a Gram-negative A component and a tripartite α-PFT complete pore are unknown and a mechanism for pore formation is still uncertain. Here we characterise the tripartite SmhABC toxin from S. marcescens and propose a mechanism of pore assembly. We present the structure of soluble SmhA, as well as the soluble and pore forms of SmhB. We show that the β-tongue soluble structure is well conserved in the family and propose two conserved latches between the head and tail domains that are broken on the soluble to pore conformational change. Using the structures of individual components, sequence analysis and docking predictions we illustrate how the A, B and C protomers would assemble on the membrane to produce a complete tripartite α-PFT pore.	Mar 2021
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Identification and structural analysis of the tripartite α-pore forming toxin of Aeromonas hydrophila Jason S. Wilson , Alicia M. Churchill-Angus , Simon P. Davies , Svetlana E. Sedelnikova , Svetomir B. Tzokov , John B. Rafferty , Per A. Bullough , Claudine Bisson , Patrick J. Baker Nature Communications 10, 2900 DOI: 10.1038/s41467-019-10777-x Diamond Proposal Number(s): [8987, 12788, 17773] Open Access Show Abstract ▼ Abstract: The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.	Jul 2019
I02-Macromolecular Crystallography	A new mechanism for high-affinity uptake of C4-dicarboxylates in bacteria revealed by the structure of rhodopseudomonas palustris matc (RPA3494), a periplasmic binding-protein of the tripartite tricarboxylate transporter (TTT) family Leonardo T. Rosa , Samuel R. Dix , John B. Rafferty , David J. Kelly Journal Of Molecular Biology DOI: 10.1016/j.jmb.2018.11.016 Diamond Proposal Number(s): [12788] Open Access Show Abstract ▼ Abstract: C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric-acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the Tripartite Tricarboxylate Transport (TTT) family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the periplasmic binding protein from this system, binds to L- and D-malate with Kd values of 27 and 21 nM respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (Kd 110 nM) and fumarate (Kd 400 nM). The 2.1 Å crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the protein structure. The substrate coordination in MatC correlates with the binding data, and explains the protein's selectivity for different substrates and respective binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized TTT family, which are widely distributed in bacterial genomes but for which details of structure–function relationships and transport mechanisms have been lacking.	Nov 2018
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis for high-affinity adipate binding to AdpC (RPA4515), an orphan periplasmic binding protein from the Tripartite Tricarboxylate Transporter (TTT) family in Rhodopseudomonas palustris Leonardo T. Rosa , Samuel R. Dix , John B. Rafferty , David J. Kelly The Febs Journal DOI: 10.1111/febs.14304 Diamond Proposal Number(s): [12788] Show Abstract ▼ Abstract: The Tripartite Tricarboxylate Transporter (TTT) family is a poorly characterised group of prokaryotic secondary solute transport systems, which employ a periplasmic substrate binding-protein (SBP) for initial ligand recognition. The substrates of only a small number of TTT systems are known and very few SBP structures have been solved, so the mechanisms of SBP-ligand interactions in this family are not well understood. The SBP RPA4515 (AdpC) from Rhodopseudomonas palustris was found by differential scanning fluorescence and isothermal titration calorimetry to bind aliphatic dicarboxylates of a chain length of six to nine carbons, with KD values in the μM range. The highest affinity was found for the C6-dicarboxylate adipate (1,6-hexanedioate). Crystal structures of AdpC with either adipate or 2-oxoadipate bound revealed a lack of positively charged amino-acids in the binding pocket and showed that water molecules are involved in bridging hydrogen bonds to the substrate, a conserved feature in the TTT SBP family that is distinct from other types of SBP. In AdpC, both of the ligand carboxylate groups and a linear chain conformation are needed for coordination in the binding pocket. RT-PCR showed that adpC expression is upregulated by low environmental adipate concentrations, suggesting adipate is a physiologically relevant substrate but as adpC is not genetically linked to any TTT membrane transport genes, the role of AdpC may be in signalling rather than transport. Our data expands the known ligands for TTT systems and identifies a novel high-affinity binding-protein for adipate, an important industrial chemical intermediate and food additive.	Oct 2017
I03-Macromolecular Crystallography	Structural insights into dynamics of RecU–HJ complex formation elucidates key role of NTR and stalk region toward formation of reactive state Sagar Khavnekar , Sarath Chandra Dantu , Svetlana Sedelnikova , Sylvia Ayora , John Rafferty , Avinash Kale Nucleic Acids Research DOI: 10.1093/nar/gkw1165 Diamond Proposal Number(s): [300] Open Access Show Abstract ▼ Abstract: Holliday junction (HJ) resolving enzyme RecU is involved in DNA repair and recombination. We have determined the crystal structure of inactive mutant (D88N) of RecU from Bacillus subtilis in complex with a 12 base palindromic DNA fragment at a resolution of 3.2 Å. This structure shows the stalk region and the essential N-terminal region (NTR) previously unseen in our DNA unbound structure. The flexible nature of the NTR in solution was confirmed using SAXS. Thermofluor studies performed to assess the stability of RecU in complex with the arms of an HJ indicate that it confers stability. Further, we performed molecular dynamics (MD) simulations of wild type and an NTR deletion variant of RecU, with and without HJ. The NTR is observed to be highly flexible in simulations of the unbound RecU, in agreement with SAXS observations. These simulations revealed domain dynamics of RecU and their role in the formation of complex with HJ. The MD simulations also elucidate key roles of the NTR, stalk region, and breathing motion of RecU in the formation of the reactive state.	Nov 2016
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography Data acquisition Diagnostics Health Physics	Direct observation of DNA threading in flap endonuclease complexes Faizah Almalki , Claudia Flemming , Jing Zhang , Min Feng , Svetlana E Sedelnikova , Tom Ceska , J B Rafferty , Jon Sayers , P J Artymiuk Nature Structural & Molecular Biology 23, 640 - 646 DOI: 10.1038/nsmb.3241 Show Abstract ▼ Abstract: Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9–2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism.	Jun 2016
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species Chatchawal Phansopa , Sumita Roy , J B Rafferty , C. W. ian Douglas , Jagroop Pandhal , Phillip C. Wright , David J Kelly , Graham p. Stafford Biochemical Journal 458, 499 - 511 DOI: 10.1042/BJ20131415 PMID: 24351045 Diamond Proposal Number(s): [8987, 7423] Open Access Show Abstract ▼ Abstract: Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), WHERE NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function. We also show that nanU is required for maximal function of the transport system and that it is expressed in a sialic acid-responsive manner. We also show its cellular localization to the outer membrane using fractionation and immunofluorescence experiments. Ligand-binding studies revealed high-affinity binding of sialic acid to NanU (Kd ~400 nM) from two Bacteroidetes species as well as binding of a range of sialic acid analogues. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure containing a novel motif characterized by an extended kinked helix that might determine sugar-binding specificity. The results of the present study characterize the first bacterial extracellular sialic acid-binding protein and define a sialic acid-specific PUL (polysaccharide utilization locus).	Mar 2014
I02-Macromolecular Crystallography	Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry Victoria Green , Fiona A. Curtis , Svetlana Sedelnikova , John B. Rafferty , Gary J. Sharples Molecular Microbiology DOI: 10.1111/mmi.12343 PMID: 23888987 Open Access Show Abstract ▼ Abstract: Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively‐charged residues in the outer sides of the junction binding cleft. Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday junction and replication fork cleavage in vivo. Two mutants, R121A and R124A that flank the DNA binding site were purified and exhibited reduced in vitro binding to fork and linear duplex substrates relative to the wild‐type, while retaining the ability to bind X junctions. Crucially, these two variants cleaved Holliday junctions with enhanced specificity and symmetry, a feature more akin to cellular RuvC resolvases. Thus, additional positive charges in the phage RuvC binding site apparently stabilize productive interactions with branched structures other than the canonical Holliday junction, a feature advantageous for viral DNA processing but deleterious for their cellular counterparts.	Jul 2013

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