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37 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4 [Next/Last]

Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS I22-Small angle scattering & Diffraction	Enterohaemorrhagic Escherichia coli AdhE spirosome length correlates with enzymatic directionality and is perturbed by salicylidene acylhydrazides Ester Serrano , Tianxiao Zhao , David R. Mark , Mostafa Soroor , Iris Floria , Nicholas J. Terrill , Nikil Kapur , Arwen I. I. Tyler , Mathew H. Horrocks , Andrew J. Roe , Olwyn Byron Open Biology 15 DOI: 10.1098/rsob.250041 Diamond Proposal Number(s): [28516] Open Access Show Abstract ▼ Abstract: Enterohaemorrhagic Escherichia coli causes sporadic, and sometimes large-scale, food poisoning outbreaks, for which antibiotic treatment in humans is contraindicated. As an alternative form of therapy, previous studies developed the family of salicylidene acylhydrazide (SA) anti-virulence compounds. One target of the SA compounds is AdhE, an enzyme that converts acetyl-CoA to ethanol and vice versa. AdhE oligomerizes, forming helicoidal filaments, heterogeneous in length, called spirosomes. We show it is possible to only partially fractionate AdhE spirosomes because in vitro they oligomerize in the absence of stimuli, and that spirosome formation is necessary to regulate the direction of AdhE enzymatic reactions. We also show that the SA compound ME0054 binds and perturbs AdhE spirosomes, enhancing the conversion of ethanol to acetyl-CoA. This mechanistic understanding of how ME0054 impacts AdhE function will help in the development of SA compounds as novel anti-virulence inhibitors.	Jun 2025
I03-Macromolecular Crystallography I23-Long wavelength MX I24-Microfocus Macromolecular Crystallography Krios II-Titan Krios II at Diamond	Structure of WzxE the lipid III flippase for Enterobacterial Common Antigen polysaccharide Audrey Le Bas , Bradley R. Clarke , Tanisha Teelucksingh , Micah Lee , Kamel El Omari , Andrew M. Giltrap , Stephen A. Mcmahon , Hui Liu , John H. Beale , Vitaliy Mykhaylyk , Ramona Duman , Neil G. Paterson , Philip N. Ward , Peter J. Harrison , Miriam Weckener , Els Pardon , Jan Steyaert , Huanting Liu , Andrew Quigley , Benjamin G. Davis , Armin Wagner , Chris Whitfield , James H. Naismith Open Biology 15 DOI: 10.1098/rsob.240310 Diamond Proposal Number(s): [33941] Open Access Show Abstract ▼ Abstract: The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.	Jan 2025
I04-Macromolecular Crystallography	The structure of an amyloid precursor protein/talin complex indicates a mechanical basis of Alzheimer’s disease Charles Ellis , Natasha L. Ward , Matthew Rice , Neil J. Ball , Pauline Walle , Chloé Najdek , Devrim Kilinc , Jean-Charles Lambert , Julien Chapuis , Benjamin T. Goult Open Biology 14 DOI: 10.1098/rsob.240185 Diamond Proposal Number(s): [28866] Open Access Show Abstract ▼ Abstract: Misprocessing of amyloid precursor protein (APP) is one of the major causes of Alzheimer’s disease. APP comprises a large extracellular region, a single transmembrane helix and a short cytoplasmic tail containing an NPxY motif (normally referred to as the YENPTY motif). Talins are synaptic scaffold proteins that connect the cytoskeletal machinery to the plasma membrane via binding NPxY motifs in the cytoplasmic tail of integrins. Here, we report the crystal structure of an APP/talin1 complex identifying a new way to couple the cytoskeletal machinery to synaptic sites through APP. Proximity ligation assay (PLA) confirmed the close proximity of talin1 and APP in primary neurons, and talin1 depletion had a dramatic effect on APP processing in cells. Structural modelling reveals APP might form an extracellular meshwork that mechanically couples the cytoskeletons of the pre- and post-synaptic compartments. We propose APP processing represents a mechanical signalling pathway whereby under tension, the cleavage sites in APP have varying accessibility to cleavage by secretases. This leads us to propose a new hypothesis for Alzheimer’s, where misregulated APP dynamics result in loss of the mechanical integrity of the synapse, corruption and loss of mechanical binary data, and excessive generation of toxic plaque-forming Aβ42 peptide.	Nov 2024
I03-Macromolecular Crystallography	Kinetoplastid kinetochore proteins KKT14–KKT15 are divergent Bub1/BubR1–Bub3 proteins Daniel Ballmer , William Carter , Jolien J. E. Van Hooff , Eelco C. Tromer , Midori Ishii , Patryk Ludzia , Bungo Akiyoshi Open Biology 14 DOI: 10.1098/rsob.240025 Diamond Proposal Number(s): [23459] Open Access Show Abstract ▼ Abstract: Faithful transmission of genetic material is crucial for the survival of all organisms. In many eukaryotes, a feedback control mechanism called the spindle checkpoint ensures chromosome segregation fidelity by delaying cell cycle progression until all chromosomes achieve proper attachment to the mitotic spindle. Kinetochores are the macromolecular complexes that act as the interface between chromosomes and spindle microtubules. While most eukaryotes have canonical kinetochore proteins that are widely conserved, kinetoplastids such as Trypanosoma brucei have a seemingly unique set of kinetochore proteins including KKT1–25. It remains poorly understood how kinetoplastids regulate cell cycle progression or ensure chromosome segregation fidelity. Here, we report a crystal structure of the C-terminal domain of KKT14 from Apiculatamorpha spiralis and uncover that it is a pseudokinase. Its structure is most similar to the kinase domain of a spindle checkpoint protein Bub1. In addition, KKT14 has a putative ABBA motif that is present in Bub1 and its paralogue BubR1. We also find that the N-terminal part of KKT14 interacts with KKT15, whose WD40 repeat beta-propeller is phylogenetically closely related to a direct interactor of Bub1/BubR1 called Bub3. Our findings indicate that KKT14–KKT15 are divergent orthologues of Bub1/BubR1–Bub3, which promote accurate chromosome segregation in trypanosomes.	Jun 2024
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural and functional characterization of nanobodies that neutralize Omicron variants of SARS-CoV-2 Katy Cornish , Jiandong Huo , Luke Jones , Parul Sharma , Joseph W. Thrush , Sahar Abdelkarim , Anja Kipar , Siva Ramadurai , Miriam Weckener , Halina Mikolajek , Sai Liu , Imogen Buckle , Eleanor Bentley , Adam Kirby , Ximeng Han , Stephen M. Laidlaw , Michelle Hill , Lauren Eyssen , Chelsea Norman , Audrey Le Bas , John Clarke , William James , James P. Stewart , Miles Carroll , James Naismith , Raymond J. Owens Open Biology 14 DOI: 10.1098/rsob.230252 Diamond Proposal Number(s): [27031] Open Access Show Abstract ▼ Abstract: The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the ‘arms race’ with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.	Jun 2024
I04-Macromolecular Crystallography	A nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation Selena G. Burgess , Nikki R. Paul , Mark W. Richards , James R. Ault , Laurie Askenatzis , Sophie G. Claydon , Ryan Corbyn , Laura M. Machesky , Richard Bayliss Open Biology 14 DOI: 10.1098/rsob.230376 Diamond Proposal Number(s): [19248] Open Access Show Abstract ▼ Abstract: Fascin-1-mediated actin-bundling activity is central to the generation of plasma membrane protrusions required for cell migration. Dysregulated formation of cellular protrusions is observed in metastatic cancers, where they are required for increased invasiveness, and is often correlated with increased Fascin-1 abundance. Therefore, there is interest in generating therapeutic Fascin-1 inhibitors. We present the identification of Nb 3E11, a nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation. The crystal structure of the Fascin-1/Nb 3E11 complex reveals the structural mechanism of inhibition. Nb 3E11 occludes an actin-binding site on the third β-trefoil domain of Fascin-1 that is currently not targeted by chemical inhibitors. Binding of Nb 3E11 to Fascin-1 induces a conformational change in the adjacent domains to stabilize Fascin-1 in an inhibitory state similar to that adopted in the presence of small-molecule inhibitors. Nb 3E11 could be used as a tool inhibitor molecule to aid in the development of Fascin-1 targeted therapeutics.	Mar 2024
I03-Macromolecular Crystallography	The structural basis of the talin-KANK1 interaction that coordinates the actin and microtubule cytoskeletons at focal adhesions Xingchen Li , Benjamin T. Goult , Christoph Ballestrem , Thomas Zacharchenko Open Biology 13 DOI: 10.1098/rsob.230058 Open Access Show Abstract ▼ Abstract: Adhesion between cells and the extracellular matrix is mediated by heterodimeric (αβ) integrin receptors that are intracellularly linked to the contractile actomyosin machinery. One of the proteins that control this link is talin, which organizes cytosolic signalling proteins into discrete complexes on β-integrin tails referred to as focal adhesions (FAs). The adapter protein KANK1 binds to talin in the region of FAs known as the adhesion belt. Here, we adapted a non-covalent crystallographic chaperone to resolve the talin–KANK1 complex. This structure revealed that the talin binding KN region of KANK1 contains a novel motif where a β-hairpin stabilizes the α-helical region, explaining both its specific interaction with talin R7 and high affinity. Single point mutants in KANK1 identified from the structure abolished the interaction and enabled us to examine KANK1 enrichment in the adhesion belt. Strikingly, in cells expressing a constitutively active form of vinculin that keeps the FA structure intact even in the presence of myosin inhibitors, KANK1 localizes throughout the entire FA structure even when actomyosin tension is released. We propose a model whereby actomyosin forces on talin eliminate KANK1 from talin binding in the centre of FAs while retaining it at the adhesion periphery.	Jun 2023
I03-Macromolecular Crystallography	PK1 from Drosophila obscurin is an inactive pseudokinase with scaffolding properties Thomas Zacharchenko , Till Dorendorf , Nicolas Locker , Evert Van Dijk , Anja Katzemich , Kay Diederichs , Belinda Bullard , Olga Mayans Open Biology 13 DOI: 10.1098/rsob.220350 Diamond Proposal Number(s): [8997] Open Access Show Abstract ▼ Abstract: Obscurins are large filamentous proteins with crucial roles in the assembly, stability and regulation of muscle. Characteristic of these proteins is a tandem of two C-terminal kinase domains, PK1 and PK2, that are separated by a long intrinsically disordered sequence. The significance of this conserved domain arrangement is unknown. Our study of PK1 from Drosophila obscurin shows that this is a pseudokinase with features typical of the CAM-kinase family, but which carries a minimalistic regulatory tail that no longer binds calmodulin or has mechanosensory properties typical of other sarcomeric kinases. PK1 binds ATP with high affinity, but in the absence of magnesium and lacks detectable phosphotransfer activity. It also has a highly diverged active site, strictly conserved across arthropods, that might have evolved to accommodate an unconventional binder. We find that PK1 interacts with PK2, suggesting a functional relation to the latter. These findings lead us to speculate that PK1/PK2 form a pseudokinase/kinase dual system, where PK1 might act as an allosteric regulator of PK2 and where mechanosensing properties, akin to those described for regulatory tails in titin-like kinases, might now reside on the unstructured interkinase segment. We propose that the PK1-interkinase-PK2 region constitutes an integrated functional unit in obscurin proteins.	Apr 2023
Krios I-Titan Krios I at Diamond	The lipid linked oligosaccharide polymerase Wzy and its regulating co-polymerase, Wzz, from enterobacterial common antigen biosynthesis form a complex Miriam Weckener , Laura S. Woodward , Bradley R. Clarke , Huanting Liu , Philip N. Ward , Audrey Le Bas , David Bhella , Chris Whitfield , James H. Naismith Open Biology 13 DOI: 10.1098/rsob.220373 Diamond Proposal Number(s): [16637] Open Access Show Abstract ▼ Abstract: The enterobacterial common antigen (ECA) is a carbohydrate polymer that is associated with the cell envelope in the Enterobacteriaceae. ECA contains a repeating trisaccharide which is polymerized by WzyE, a member of the Wzy membrane protein polymerase superfamily. WzyE activity is regulated by a membrane protein polysaccharide co-polymerase, WzzE. Förster resonance energy transfer experiments demonstrate that WzyE and WzzE from Pectobacterium atrosepticum form a complex in vivo, and immunoblotting and cryo-electron microscopy (cryo-EM) analysis confirm a defined stoichiometry of approximately eight WzzE to one WzyE. Low-resolution cryo-EM reconstructions of the complex, aided by an antibody recognizing the C-terminus of WzyE, reveals WzyE sits in the central membrane lumen formed by the octameric arrangement of the transmembrane helices of WzzE. The pairing of Wzy and Wzz is found in polymerization systems for other bacterial polymers, including lipopolysaccharide O-antigens and capsular polysaccharides. The data provide new structural insight into a conserved mechanism for regulating polysaccharide chain length in bacteria.	Mar 2023
I03-Macromolecular Crystallography	Experimental and computational snapshots of C-C bond formation in a C-nucleoside synthase Wenbo Li , Georgina C. Girt , Ashish Radadiya , James J. P. Stewart , Nigel G. J. Richards , James H. Naismith Open Biology 13 DOI: 10.1098/rsob.220287 Open Access Show Abstract ▼ Abstract: The biosynthetic enzyme, ForT, catalyses the formation of a C-C bond between 4-amino-1H-pyrazoledicarboxylic acid and MgPRPP to produce a C-nucleoside precursor of formycin A. The transformation catalysed by ForT is of chemical interest because it is one of only a few examples in which C-C bond formation takes place via an electrophilic substitution of a small, aromatic heterocycle. In addition, ForT is capable of discriminating between the aminopyrazoledicarboxylic acid and an analogue in which the amine is replaced by a hydroxyl group; a remarkable feat given the steric and electronic similarities of the two molecules. Here we report biophysical measurements, structural biology and quantum chemical calculations that provide a detailed molecular picture of ForT-catalysed C-C bond formation and the conformational changes that are coupled to catalysis. Our findings set the scene for employing engineered ForT variants in the biocatalytic production of novel, anti-viral C-nucleoside and C-nucleotide analogues.	Jan 2023

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