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1,245 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6, 7, 8 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength) I23-Long wavelength MX	Novel quaternary structures of the human prion protein globular domain Leandro Oliveira Bortot , Victor Lopes Rangel , Francesca A. Pavlovici , Kamel El Omari , Armin Wagner , Jose Brandao-Neto , Romain Talon , Frank Von Delft , Andrew G. Reidenbach , Sonia M. Vallabh , Eric Vallabh Minikel , Stuart Schreiber , Maria Cristina Nonato Biochimie 191, 118 - 125 DOI: 10.1016/j.biochi.2021.09.005 Diamond Proposal Number(s): [18954] Show Abstract ▼ Abstract: Prion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process.	Dec 2021
I24-Microfocus Macromolecular Crystallography	Experimental evidence for the benefits of higher X-ray energies for macromolecular crystallography Selina L. S. Storm , Danny Axford , Robin L. Owen Iucrj 8 DOI: 10.1107/S2052252521008423 Open Access Show Abstract ▼ Abstract: X-ray-induced radiation damage is a limiting factor for the macromolecular crystallographer and data must often be merged from many crystals to yield complete data sets for the structure solution of challenging samples. Increasing the X-ray energy beyond the typical 10–15 keV range promises to provide an extension of crystal lifetime via an increase in diffraction efficiency. To date, however, hardware limitations have negated any possible gains. Through the first use of a cadmium telluride EIGER2 detector and a beamline optimized for high-energy data collection, it is shown that at higher energies fewer crystals will be required to obtain complete data, as the diffracted intensity per unit dose increases by a factor of more than two between 12.4 and 25 keV. Additionally, these higher energy data can provide more information, as shown by a systematic increase in the high-resolution cutoff of the data collected. Taken together, these gains point to a high-energy future for synchrotron-based macromolecular crystallography.	Nov 2021
I03-Macromolecular Crystallography	Identification of unprecedented ATP-competitive choline kinase inhibitors Francesca Quartieri , Marcella Nesi , Nilla R. Avanzi , Daniela Borghi , Elena Casale , Emiliana Corti , Ulisse Cucchi , Daniele Donati , Marina Fasolini , Eduard R. Felder , Arturo Galvani , Maria L. Giorgini , Antonio Lomolino , Maria Menichincheri , Christian Orrenius , Claudia Perrera , Stefania Re Depaolini , Federico Riccardi-Sirtori , Enea Salsi , Antonella Isacchi , Paola Gnocchi Bioorganic & Medicinal Chemistry Letters 51 DOI: 10.1016/j.bmcl.2021.128310 Diamond Proposal Number(s): [26586] Show Abstract ▼ Abstract: In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.	Nov 2021
I03-Macromolecular Crystallography	Regulation of CYLD activity and specificity by phosphorylation and ubiquitin-binding CAP-Gly domains Paul R. Elliott , Derek Leske , Jane Wagstaff , Lisa Schlicher , Georgina Berridge , Sarah Maslen , Frederik Timmermann , Biao Ma , Roman Fischer , Stefan M. V. Freund , David Komander , Mads Gyrd-Hansen Cell Reports 37 DOI: 10.1016/j.celrep.2021.109777 Open Access Show Abstract ▼ Abstract: Non-degradative ubiquitin chains and phosphorylation events govern signaling responses by innate immune receptors. The deubiquitinase CYLD in complex with SPATA2 is recruited to receptor signaling complexes by the ubiquitin ligase LUBAC and regulates Met1- and Lys63-linked polyubiquitin and receptor signaling outcomes. Here, we investigate the molecular determinants of CYLD activity. We reveal that two CAP-Gly domains in CYLD are ubiquitin-binding domains and demonstrate a requirement of CAP-Gly3 for CYLD activity and regulation of immune receptor signaling. Moreover, we identify a phosphorylation switch outside of the catalytic USP domain, which activates CYLD toward Lys63-linked polyubiquitin. The phosphorylated residue Ser568 is a novel tumor necrosis factor (TNF)-regulated phosphorylation site in CYLD and works in concert with Ser418 to enable CYLD-mediated deubiquitination and immune receptor signaling. We propose that phosphorylated CYLD, together with SPATA2 and LUBAC, functions as a ubiquitin-editing complex that balances Lys63- and Met1-linked polyubiquitin at receptor signaling complexes to promote LUBAC signaling.	Oct 2021
I24-Microfocus Macromolecular Crystallography	Structural basis for silicic acid uptake by higher plants Bert Van Den Berg , Conrado Pedebos , Jani R. Bolla , Carol V. Robinson , Arnaud Basle , Syma Khalid Journal Of Molecular Biology 433 DOI: 10.1016/j.jmb.2021.167226 Open Access Show Abstract ▼ Abstract: Many of the world's most important food crops such as rice, barley and maize accumulate silicon (Si) to high levels, resulting in better plant growth and crop yields. The first step in Si accumulation is the uptake of silicic acid by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins, also named metalloid porins. Here, we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). The OsNIP2;1 channel is closed in the crystal structure by the cytoplasmic loop D, which is known to regulate channel opening in classical plant aquaporins. The structure further reveals a novel, five-residue extracellular selectivity filter with a large diameter. Unbiased molecular dynamics simulations show a rapid opening of the channel and visualise how silicic acid interacts with the selectivity filter prior to transmembrane diffusion. Our results will enable detailed structure–function studies of metalloid porins, including the basis of their substrate selectivity.	Oct 2021
I24-Microfocus Macromolecular Crystallography	Crystallography and QM/MM simulations identify preferential binding of hydrolyzed carbapenem and penem antibiotics to the l1 metallo-β-lactamase in the imine form Rebecca M. Twidale , Philip Hinchliffe , James Spencer , Adrian J. Mulholland Journal Of Chemical Information And Modeling DOI: 10.1021/acs.jcim.1c00663 Diamond Proposal Number(s): [17212] Show Abstract ▼ Abstract: Widespread bacterial resistance to carbapenem antibiotics is an increasing global health concern. Resistance has emerged due to carbapenem-hydrolyzing enzymes, including metallo-β-lactamases (MβLs), but despite their prevalence and clinical importance, MβL mechanisms are still not fully understood. Carbapenem hydrolysis by MβLs can yield alternative product tautomers with the potential to access different binding modes. Here, we show that a combined approach employing crystallography and quantum mechanics/molecular mechanics (QM/MM) simulations allow tautomer assignment in MβL:hydrolyzed antibiotic complexes. Molecular simulations also examine (meta)stable species of alternative protonation and tautomeric states, providing mechanistic insights into β-lactam hydrolysis. We report the crystal structure of the hydrolyzed carbapenem ertapenem bound to the L1 MβL from Stenotrophomonas maltophilia and model alternative tautomeric and protonation states of both hydrolyzed ertapenem and faropenem (a related penem antibiotic), which display different binding modes with L1. We show how the structures of both complexed β-lactams are best described as the (2S)-imine tautomer with the carboxylate formed after β-lactam ring cleavage deprotonated. Simulations show that enamine tautomer complexes are significantly less stable (e.g., showing partial loss of interactions with the L1 binuclear zinc center) and not consistent with experimental data. Strong interactions of Tyr32 and one zinc ion (Zn1) with ertapenem prevent a C6 group rotation, explaining the different binding modes of the two β-lactams. Our findings establish the relative stability of different hydrolyzed (carba)penem forms in the L1 active site and identify interactions important to stable complex formation, information that should assist inhibitor design for this important antibiotic resistance determinant.	Oct 2021
I04-Macromolecular Crystallography	Structure of the fungal hydroxylase, CYP505A30, and rational transfer of mutation data from CYP102A1 to alter regioselectivity Jasmin Cara Aschenbrenner , Ana C. Ebrecht , Carmien Tolmie , Martha Sophia Smit , Diederik J. Opperman Catalysis Science & Technology DOI: 10.1039/D1CY01348C Diamond Proposal Number(s): [20303] Open Access Show Abstract ▼ Abstract: CYP505A30 is a fungal, self-sufficient cytochrome P450 monooxygenase that can selectively oxyfunctionalise n-alkanes, fatty alcohols, and fatty acids. From alkanes, it produces a mixture of non-vicinal diols by two sequential hydroxylation reactions. Here we report the structure of the haem domain of CYP505A30, the first structure for a member of the CYP505 family, with dodecanoic acid bound within the active site. Overall, a high structural similarity to the related bacterial CYP102A1 was observed, despite low sequence identity (<40 %). Comparison of the active sites, however, showed a high degree of conservation with only two amino acid differences close to the haem. Stabilisation of the acid substrate in CYP505A30 also occurs, as in CYP102A1, via an arginine residue. However, compared to R47, which is situated in the β1 region of CYP102A1, R358 is located in the β3 region of CYP505A30. We furthermore created mutants to test if it is possible to rationally transfer the knowledge on active site mutations in CYP102A1 to change the regioselectivity of CYP505A30. The introduction of F93V, I334F mutations resulted in increased ω-1 (C2) regioselectivity, similar to CYP102A1 328-87, of more than 80 % for n-octane and 90 % for n-decane. Changing residues to resemble the 102A1 wildtype increased the regioselectivity towards ω-2 (C3) to over 60 % for both substrates. The knowledge gained from this study unlocks a more selective production of symmetrical non-vicinal diols from n-alkanes.	Oct 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Inverting the stereoselectivity of an NADH‐dependent imine‐reductase variant Peter Stockinger , Niels Borlinghaus , Mahima Sharma , Benjamin Aberle , Gideon Grogan , Jürgen Pleiss , Bettina M. Nestl Chemcatchem DOI: 10.1002/cctc.202101057 Diamond Proposal Number(s): [9948] Open Access Show Abstract ▼ Abstract: Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the ( R )-selective IRED from Myxococcus stipitatus (( R )-IRED- Ms _V8) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and ( S )-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of ( R )-IRED- Ms _V8. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2-methylpyrroline compared to the wild-type and afforded the ( S )-amine product with >99% conversion and 91% enantiomeric excess. We also report the crystal-structures of the NADPH-dependent ( R )-IRED- Ms wild-type enzyme and the NADH-dependent ( R )-IRED- Ms _V8 variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.	Oct 2021
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	The phage defence island of a multidrug resistant plasmid uses both BREX and type IV restriction for complementary protection from viruses David M. Picton , Yvette A. Luyten , Richard D. Morgan , Darren L. Smith , David T. F. Dryden , Jay C. D. Hinton , Tim R. Blower Nucleic Acids Research DOI: 10.1093/nar/gkab906 Diamond Proposal Number(s): [24948] Open Access Show Abstract ▼ Abstract: Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in ‘defence islands’, providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.	Oct 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	GenoChemetic strategy for derivatization of the violacein natural product scaffold Hung-En Lai , Alan M. C. Obled , Soo Mei Chee , Rhodri M. Morgan , Rosemary Lynch , Sunil V. Sharma , Simon J. Moore , Karen M. Polizzi , Rebecca J. M. Goss , Paul S. Freemont Acs Chemical Biology 25 DOI: 10.1021/acschembio.1c00483 Diamond Proposal Number(s): [12579] Open Access Show Abstract ▼ Abstract: Natural products and their analogues are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds, which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, six halogenated analogues of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein analogues via the Suzuki–Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein we demonstrate a flexible and rapid approach to access a diverse chemical space that can be applied to a wide range of natural product scaffolds.	Oct 2021

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