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

Instruments / Technical Area	Publication Details	Published
Krios II-Titan Krios II at Diamond Krios IV-Titan Krios IV at Diamond	Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2 Huanyu Z. Li , Ashley C. Pike , Yung-Ning Chang , Dheeraj Prakaash , Zuzana Gelova , Josefina Stanka , Christophe Moreau , Hannah C. Scott , Frank Wunder , Gernot Wolf , Andreea Scacioc , Gavin Mckinley , Helena Batoulis , Shubhashish Mukhopadhyay , Andrea Garofoli , Adán Pinto-Fernández , Benedikt M. Kessler , Nicola A. Burgess-Brown , Saša Štefanić , Tabea Wiedmer , Katharina L. Duerr , Vera Puetter , Alexander Ehrmann , Syma Khalid , Alvaro Ingles-Prieto , Giulio Superti-Furga , David B. Sauer Nature Communications 16 DOI: 10.1038/s41467-025-55942-7 Diamond Proposal Number(s): [28713] Open Access Show Abstract ▼ Abstract: Sphingosine-1-phosphate (S1P) is a signaling lysolipid critical to heart development, immunity, and hearing. Accordingly, mutations in the S1P transporter SPNS2 are associated with reduced white cell count and hearing defects. SPNS2 also exports the S1P-mimicking FTY720-P (Fingolimod) and thereby is central to the pharmacokinetics of this drug when treating multiple sclerosis. Here, we use a combination of cryo-electron microscopy, immunofluorescence, in vitro binding and in vivo S1P export assays, and molecular dynamics simulations to probe SPNS2’s substrate binding and transport. These results reveal the transporter’s binding mode to its native substrate S1P, the therapeutic FTY720-P, and the reported SPNS2-targeting inhibitor 33p. Further capturing an inward-facing apo state, our structures illuminate the protein’s mechanism for exchange between inward-facing and outward-facing conformations. Finally, using these structural, localization, and S1P transport results, we identify how pathogenic mutations ablate the protein’s export activity and thereby lead to hearing loss.	Jan 2025
I04-1-Macromolecular Crystallography (fixed wavelength)	Conformational plasticity of the ULK3 kinase domain Sebastian Mathea , Eidarus Salah , Cynthia Tallant , Deep Chatterjee , Benedict-Tilman Berger , Rebecca Konietzny , Susanne Müller-Knapp , Benedikt M Kessler , Stefan Knapp Biochemical Journal DOI: 10.1042/BCJ20210257 Open Access Show Abstract ▼ Abstract: The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein.	Jun 2021
	Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19 Yanchun Peng , Alexander J. Mentzer , Guihai Liu , Xuan Yao , Zixi Yin , Danning Dong , Wanwisa Dejnirattisai , Timothy Rostron , Piyada Supasa , Chang Liu , César López-Camacho , Jose Slon-Campos , Yuguang Zhao , David I. Stuart , Guido C. Paesen , Jonathan M. Grimes , Alfred A. Antson , Oliver W. Bayfield , Dorothy E. D. P. Hawkins , De-Sheng Ker , Beibei Wang , Lance Turtle , Krishanthi Subramaniam , Paul Thomson , Ping Zhang , Christina Dold , Jeremy Ratcliff , Peter Simmonds , Thushan De Silva , Paul Sopp , Dannielle Wellington , Ushani Rajapaksa , Yi-Ling Chen , Mariolina Salio , Giorgio Napolitani , Wayne Paes , Persephone Borrow , Benedikt M. Kessler , Jeremy W. Fry , Nikolai F. Schwabe , Malcolm G. Semple , J. Kenneth Baillie , Shona C. Moore , Peter J. M. Openshaw , M. Azim Ansari , Susanna Dunachie , Eleanor Barnes , John Frater , Georgina Kerr , Oliver Gould , Teresa Lockett , Robert Levin , Yonghong Zhang , Ronghua Jing , Ling-Pei Ho , Richard J. Cornall , Christopher P. Conlon , Paul Klenerman , Gavin R. Screaton , Juthathip Mongkolsapaya , Andrew Mcmichael , Julian C. Knight , Graham Ogg , Tao Dong Nature Immunology 7 DOI: 10.1038/s41590-020-0782-6 Open Access Show Abstract ▼ Abstract: The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide–MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.	Sep 2020
I03-Macromolecular Crystallography	Regulation of the endosomal SNX27-retromer by OTULIN Aurelia Stangl , Paul R. Elliott , Adan Pinto-Fernandez , Sarah Bonham , Luke Harrison , Annalisa Schaub , Kerstin Kutzner , Kirstin Keusekotten , Paul T. Pfluger , Farid El Oualid , Benedikt M. Kessler , David Komander , Daniel Krappmann Nature Communications 10 DOI: 10.1038/s41467-019-12309-z Open Access Show Abstract ▼ Abstract: OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association, OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the VPS26A-retromer subunit and endosome-to-plasma membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of SNX27-retromer assembly and recycling to the cell surface.	Sep 2019
I03-Macromolecular Crystallography	An activity-based probe targeting non-catalytic, highly conserved amino acid residues within bromodomains Melissa D'Ascenzio , Kathryn M. Pugh , Rebecca Konietzny , Georgina Berridge , Cynthia Tallant , Shaima Hashem , Octovia Monteiro , Jason R. Thomas , Markus Schirle , Stefan Knapp , Brian Marsden , Oleg Fedorov , Chas Bountra , Benedikt M. Kessler , Paul E. Brennan Angewandte Chemie International Edition 58, 1007 - 1012 DOI: 10.1002/anie.201807825 Diamond Proposal Number(s): [10619] Open Access Show Abstract ▼ Abstract: Bromodomain‐containing proteins are epigenetic modulators involved in a wide range of cellular processes, from recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl‐lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule‐based approach to covalently modify recombinant and endogenous bromodomain‐containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag allowed in‐gel visualization and pull‐down of the desired bromodomains.	Jan 2019
I04-Macromolecular Crystallography	Design, synthesis and characterization of covalent kdm5 inhibitors Saleta Vazquez-Rodriguez , Miranda Wright , Catherine M. Rogers , Adam P. Cribbs , Srikannathasan Velupillai , Martin Philpott , Henry Lee , James E. Dunford , Kilian V. M. Huber , Matthew B. Robers , James D. Vasta , Marie-Laetitia Thezenas , Sarah Bonham , Benedikt Kessler , James Bennett , Oleg Fedorov , Florence Raynaud , Adam Donovan , Julian Blagg , Vassilios Bavetsias , Udo Oppermann , Chas Bountra , Akane Kawamura , Paul E. Brennan Angewandte Chemie International Edition 58, 515 - 519 DOI: 10.1002/anie.201810179 Diamond Proposal Number(s): [15433] Open Access Show Abstract ▼ Abstract: Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub‐family. The covalent binding to the targeted proteins was confirmed by MS and time‐dependent inhibition. Additional competition assays show that compounds were non 2‐OG competitive. Target engagement and ChIP‐seq analysis showed that the compounds inhibited the KDM5 members in cells at nano‐ to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.	Jan 2019
I04-Macromolecular Crystallography	The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases Suzana Markolovic , Qinqin Zhuang , Sarah E. Wilkins , Charlotte D. Eaton , Martine I. Abboud , Maximiliano J. Katz , Helen E. Mcneil , Robert K. Leśniak , Charlotte Hall , Weston B. Struwe , Rebecca Konietzny , Simon Davis , Ming Yang , Wei Ge , Justin L. P. Benesch , Benedikt M. Kessler , Peter J. Ratcliffe , Matthew E. Cockman , Roman Fischer , Pablo Wappner , Rasheduzzaman Chowdhury , Mathew L. Coleman , Christopher J. Schofield Nature Chemical Biology 14, 688 - 695 DOI: 10.1038/s41589-018-0071-y Show Abstract ▼ Abstract: Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(ii)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.	Jun 2018
I02-Macromolecular Crystallography	Discovery of a selective allosteric inhibitor targeting macrodomain 2 of poly-adenosine-diphosphate-ribose polymerases 14 Marion Schuller , Kerstin Riedel , Ian Gibbs-Seymour , Kristin Uth , Christian Sieg , André P Gehring , Ivan Ahel , Franz Bracher , Benedikt M. Kessler , Jonathan M. Elkins , Stefan Knapp Acs Chemical Biology DOI: 10.1021/acschembio.7b00445 Show Abstract ▼ Abstract: Macrodomains are conserved protein interaction modules that can be found in all domains of life as well as in certain viruses. Macrodomains mediate recognition of sequence motifs harbouring adenosine diphosphate ribose (ADPR) modifications, thereby regulating a variety of cellular processes. Due to their role in cancer or viral pathogenesis, macrodomains have emerged as potential therapeutic targets, but the unavailability of small molecule inhibitors has hampered target validation studies so far. Here, we describe an efficient screening strategy for identification of small molecule inhibitors that displace ADPR from macrodomains. We report the discovery and characterisation of a macrodomain inhibitor, GeA-69, selectively targeting macrodomain 2 (MD2) of PARP14 with low micromolar affinity. Co-crystallisation of a GeA-69 analogue with PARP14 MD2 revealed an allosteric binding mechanism explaining its selectivity over other human macrodomains. We show that GeA-69 engages PARP14 MD2 in intact cells and prevents its localisation to sites of DNA damage.	Oct 2017
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Molecular basis of USP7 inhibition by selective small-molecule inhibitors Andrew Turnbull , Stephanos Ioannidis , Wojciech W. Krajewski , Adan Pinto-Fernandez , Claire Heride , Agnes C. L. Martin , Louise M. Tonkin , Elizabeth C. Townsend , Shane M. Buker , David R. Lancia , Justin A. Caravella , Angela V. Toms , Thomas M. Charlton , Johanna Lahdenranta , Erik Wilker , Bruce C. Follows , Nicola J. Evans , Lucy Stead , Cristina Alli , Vladislav V. Zarayskiy , Adam C. Talbot , Alexandre J. Buckmelter , Minghua Wang , Crystal L. Mckinnon , Fabienne Saab , Joanna F. Mcgouran , Hannah Century , Malte Gersch , Marc S. Pittman , C. Gary Marshall , Tony M. Raynham , Mary Simcox , Lorna M. D. Stewart , Sheila B. Mcloughlin , Jaime A. Escobedo , Kenneth W. Bair , Christopher J. Dinsmore , Tim R. Hammonds , Sunkyu Kim , Sylvie Urbé , Michael J. Clague , Benedikt M. Kessler , David Komander Nature 12 DOI: 10.1038/nature24451 Diamond Proposal Number(s): [15419, 5070] Show Abstract ▼ Abstract: Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise ‘undruggable’ targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.	Oct 2017
I04-1-Macromolecular Crystallography (fixed wavelength)	A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN Aurelia Weber , Paul R. Elliott , Adan Pinto-Fernandez , Sarah Bonham , Benedikt M. Kessler , David Komander , Farid El Oualid , Daniel Krappmann Cell Chemical Biology DOI: 10.1016/j.chembiol.2017.08.006 Diamond Proposal Number(s): [15916] Open Access Show Abstract ▼ Abstract: The methionine 1 (M1)-specific deubiquitinase (DUB) OTULIN acts as a negative regulator of nuclear factor κB signaling and immune homeostasis. By replacing Gly76 in distal ubiquitin (Ub) by dehydroalanine we designed the diubiquitin (diUb) activity-based probe UbG76Dha-Ub (OTULIN activity-based probe [ABP]) that couples to the catalytic site of OTULIN and thereby captures OTULIN in its active conformation. The OTULIN ABP displays high selectivity for OTULIN and does not label other M1-cleaving DUBs, including CYLD. The only detectable cross-reactivities were the labeling of USP5 (Isopeptidase T) and an ATP-dependent assembly of polyOTULIN ABP chains via Ub-activating E1 enzymes. Both cross-reactivities were abolished by the removal of the C-terminal Gly in the ABP's proximal Ub, yielding the specific OTULIN probe UbG76Dha-UbΔG76 (OTULIN ABPΔG76). Pull-downs demonstrate that substrate-bound OTULIN associates with the linear ubiquitin chain assembly complex (LUBAC). Thus, we present a highly selective ABP for OTULIN that will facilitate studying the cellular function of this essential DUB.	Sep 2017

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