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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
	Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations C. Keith Cassidy , Benjamin A. Himes , Dapeng Sun , Jun Ma , Gongpu Zhao , John S. Parkinson , Phillip J. Stansfeld , Zaida Luthey-schulten , Peijun Zhang Communications Biology 3 DOI: 10.1038/s42003-019-0748-0 Show Abstract ▼ Abstract: To enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism.	Dec 2020
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Caffeine inhibits Notum activity by binding at the catalytic pocket Yuguang Zhao , Jingshan Ren , James Hillier , Weixian Lu , Edith Yvonne Jones Communications Biology 3 DOI: 10.1038/s42003-020-01286-5 Diamond Proposal Number(s): [14744] Open Access Show Abstract ▼ Abstract: Notum inhibits Wnt signalling via enzymatic delipidation of Wnt ligands. Restoration of Wnt signalling by small molecule inhibition of Notum may be of therapeutic benefit in a number of pathologies including Alzheimer’s disease. Here we report Notum activity can be inhibited by caffeine (IC50 19 µM), but not by demethylated caffeine metabolites: paraxanthine, theobromine and theophylline. Cellular luciferase assays show Notum-suppressed Wnt3a function can be restored by caffeine with an EC50 of 46 µM. The dissociation constant (Kd) between Notum and caffeine is 85 µM as measured by surface plasmon resonance. High-resolution crystal structures of Notum complexes with caffeine and its minor metabolite theophylline show both compounds bind at the centre of the enzymatic pocket, overlapping the position of the natural substrate palmitoleic lipid, but using different binding modes. The structural information reported here may be of relevance for the design of more potent brain-accessible Notum inhibitors.	Oct 2020
I03-Macromolecular Crystallography	Structural effects of the highly protective V127 polymorphism on human prion protein Laszlo L. P. Hosszu , Rebecca Conners , Daljit Sangar , Mark Batchelor , Elizabeth B. Sawyer , Stuart Fisher , Matthew J. Cliff , Andrea M. Hounslow , Katherine Mcauley , R. Leo Brady , Graham S. Jackson , Jan Bieschke , Jonathan P. Waltho , John Collinge Communications Biology 3 DOI: 10.1038/s42003-020-01126-6 Diamond Proposal Number(s): [4923, 5969] Open Access Show Abstract ▼ Abstract: Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease.	Jul 2020
I04-Macromolecular Crystallography	Ebselen as template for stabilization of A4V mutant dimer for motor neuron disease therapy Varunya Chantadul , Gareth S. A. Wright , Kangsa Amporndanai , Munazza Shahid , Svetlana V. Antonyuk , Gina Washbourn , Michael Rogers , Natalie Roberts , Matthew Pye , Paul M. O’neill , S. Samar Hasnain Communications Biology 3 DOI: 10.1038/s42003-020-0826-3 Diamond Proposal Number(s): [15991, 21970] Open Access Show Abstract ▼ Abstract: Mutations to the gene encoding superoxide dismutase-1 (SOD1) were the first genetic elements discovered that cause motor neuron disease (MND). These mutations result in compromised SOD1 dimer stability, with one of the severest and most common mutations Ala4Val (A4V) displaying a propensity to monomerise and aggregate leading to neuronal death. We show that the clinically used ebselen and related analogues promote thermal stability of A4V SOD1 when binding to Cys111 only. We have developed a A4V SOD1 differential scanning fluorescence-based assay on a C6S mutation background that is effective in assessing suitability of compounds. Crystallographic data show that the selenium atom of these compounds binds covalently to A4V SOD1 at Cys111 at the dimer interface, resulting in stabilisation. This together with chemical amenability for hit expansion of ebselen and its on-target SOD1 pharmacological chaperone activity holds remarkable promise for structure-based therapeutics for MND using ebselen as a template.	Mar 2020
Krios II-Titan Krios II at Diamond	Cryo-EM study of an archaeal 30S initiation complex gives insights into evolution of translation initiation Pierre-damien Coureux , Christine Lazennec-schurdevin , Sophie Bourcier , Yves Mechulam , Emmanuelle Schmitt Communications Biology 3 DOI: 10.1038/s42003-020-0780-0 Diamond Proposal Number(s): [16410] Open Access Show Abstract ▼ Abstract: Archaeal translation initiation occurs within a macromolecular complex containing the small ribosomal subunit (30S) bound to mRNA, initiation factors aIF1, aIF1A and the ternary complex aIF2:GDPNP:Met-tRNAiMet. Here, we determine the cryo-EM structure of a 30S:mRNA:aIF1A:aIF2:GTP:Met-tRNAiMet complex from Pyrococcus abyssi at 3.2 Å resolution. It highlights archaeal features in ribosomal proteins and rRNA modifications. We find an aS21 protein, at the location of eS21 in eukaryotic ribosomes. Moreover, we identify an N-terminal extension of archaeal eL41 contacting the P site. We characterize 34 N4-acetylcytidines distributed throughout 16S rRNA, likely contributing to hyperthermostability. Without aIF1, the 30S head is stabilized and initiator tRNA is tightly bound to the P site. A network of interactions involving tRNA, mRNA, rRNA modified nucleotides and C-terminal tails of uS9, uS13 and uS19 is observed. Universal features and domain-specific idiosyncrasies of translation initiation are discussed in light of ribosomal structures from representatives of each domain of life.	Feb 2020
I03-Macromolecular Crystallography	Glutathione facilitates enterovirus assembly by binding at a druggable pocket Helen M. E. Duyvesteyn , Jingshan Ren , Thomas S. Walter , Elizabeth E. Fry , David I. Stuart Communications Biology 3 DOI: 10.1038/s42003-019-0722-x Diamond Proposal Number(s): [19946] Open Access Show Abstract ▼ Abstract: Enteroviruses cause a range of human and animal diseases, some life-threatening, but there remain no licenced anti-enterovirus drugs. However, a benzene-sulfonamide derivative and related compounds have been shown recently to block infection of a range of enteroviruses by binding the capsid at a positively-charged surface depression conserved across many enteroviruses. It has also been established that glutathione is essential for the assembly of many enteroviruses, interacting with the capsid proteins to facilitate the formation of the pentameric assembly intermediate, although the mechanism is unknown. Here we show, by high resolution structure analyses of enterovirus F3, that reduced glutathione binds to the same interprotomer pocket as the benzene-sulfonamide derivative. Bound glutathione makes strong interactions with adjacent protomers, thereby explaining the underlying biological role of this druggable binding pocket and delineating the pharmacophore for potential antivirals.	Jan 2020
I04-Macromolecular Crystallography	High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity Wen Zhu , Ashish Radadiya , Claudine Bisson , Sabine Wenzel , Brian E. Nordin , Francisco Martínez-márquez , Tsuyoshi Imasaki , Svetlana E. Sedelnikova , Adriana Coricello , Patrick Baumann , Alexandria H. Berry , Tyzoon K. Nomanbhoy , John W. Kozarich , Yi Jin , David W. Rice , Yuichiro Takagi , Nigel G. J. Richards Communications Biology 2 DOI: 10.1038/s42003-019-0587-z Diamond Proposal Number(s): [12788] Open Access Show Abstract ▼ Abstract: Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.	Sep 2019
I04-Macromolecular Crystallography	High-resolution crystal structure of arthropod Eiger TNF suggests a mode of receptor engagement and altered surface charge within endosomes Mattia Bertinelli , Guido C. Paesen , Jonathan M. Grimes , Max Renner Communications Biology 2 DOI: 10.1038/s42003-019-0541-0 Diamond Proposal Number(s): [14774] Open Access Show Abstract ▼ Abstract: The tumour necrosis factor alpha (TNFα) superfamily of proteins are critical in numerous biological processes, such as in development and immunity. Eiger is the sole TNFα member described in arthropods such as in the important model organism Drosophila. To date there are no structural data on any Eiger protein. Here we present the structure of the TNF domain of Eiger from the fall armyworm Spodoptera frugiperda (SfEiger) to 1.7 Å from a serendipitously obtained crystal without prior knowledge of the protein sequence. Our structure confirms that canonical trimerization is conserved from ancestral TNFs and points towards a mode of receptor engagement. Furthermore, we observe numerous surface histidines on SfEiger, potentially acting as pH switches following internalization into endosomes. Our data contributes to the genome annotation of S. frugiperda, a voracious agricultural pest, and can serve as a basis for future structure-function investigations of the TNF system in related arthropods such as Drosophila.	Aug 2019
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	A brain-permeable inhibitor of the neurodegenerative disease target kynurenine 3-monooxygenase prevents accumulation of neurotoxic metabolites Shaowei Zhang , Michiyo Sakuma , Girdhar S. Deora , Colin Levy , Alex Klausing , Carlo Breda , Kevin D. Read , Chris D. Edlin , Benjamin P. Ross , Marina Wright Muelas , Philip J. Day , Stephen O’hagan , Douglas B. Kell , Robert Schwarcz , David Leys , Derren J. Heyes , Flaviano Giorgini , Nigel S. Scrutton Communications Biology 2 DOI: 10.1038/s42003-019-0520-5 Diamond Proposal Number(s): [8997, 12788] Open Access Show Abstract ▼ Abstract: Dysregulation of the kynurenine pathway (KP) leads to imbalances in neuroactive metabolites associated with the pathogenesis of several neurodegenerative disorders, including Huntington’s disease (HD). Inhibition of the enzyme kynurenine 3-monooxygenase (KMO) in the KP normalises these metabolic imbalances and ameliorates neurodegeneration and related phenotypes in several neurodegenerative disease models. KMO is thus a promising candidate drug target for these disorders, but known inhibitors are not brain permeable. Here, 19 new KMO inhibitors have been identified. One of these (1) is neuroprotective in a Drosophila HD model but is minimally brain penetrant in mice. The prodrug variant (1b) crosses the blood–brain barrier, releases 1 in the brain, thereby lowering levels of 3-hydroxykynurenine, a toxic KP metabolite linked to neurodegeneration. Prodrug 1b will advance development of targeted therapies against multiple neurodegenerative and neuroinflammatory diseases in which KP likely plays a role, including HD, Alzheimer’s disease, and Parkinson’s disease.	Jul 2019
Krios II-Titan Krios II at Diamond	Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure Andani Mulelu , Angela Kirykowicz , Jeremy Woodward Communications Biology 2 DOI: 10.1038/s42003-019-0505-4 PMID: 31341959 Diamond Proposal Number(s): [20975] Open Access Show Abstract ▼ Abstract: Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 Å) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from Arabidopsis thaliana. We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between α2 and α3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis.	Jul 2019

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