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

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Filamin C dimerisation is regulated by HSPB7 Zihao Wang , Guodong Cao , Miranda P. Collier , Xingyu Qiu , Sophie Broadway-Stringer , Dominik Šaman , Jediael Z. Y. Ng , Navoneel Sen , Amar J. Azad , Charlotte Hooper , Johannes Zimmermann , Michael A. Mcdonough , Jurgen Brem , Patrick Rabe , Haigang Song , T. Reid Alderson , Christopher J. Schofield , Jani R. Bolla , Kristina Djinovic-Carugo , Dieter O. Fürst , Bettina Warscheid , Matteo T. Degiacomi , Timothy M. Allison , Georg K. A. Hochberg , Carol V. Robinson , Katja Gehmlich , Justin L. P. Benesch Nature Communications 16 DOI: 10.1038/s41467-025-58889-x Diamond Proposal Number(s): [19458] Open Access Show Abstract ▼ Abstract: The biomechanical properties and responses of tissues underpin a variety important of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. FLNC is a multi-functional protein that interacts with a variety of partners, however, how it is regulated at the molecular level is not well understood. Here we investigate its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We find that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we solve by X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work therefore shows, structurally, how HSPB7 acts as a specific molecular chaperone that regulates FLNC dimerisation.	May 2025
I03-Macromolecular Crystallography	Biochemical and crystallographic studies of l,d-transpeptidase 2 from Mycobacterium tuberculosis with its natural monomer substrate Mariska De Munnik , Pauline A. Lang , Karina Calvopina , Patrick Rabe , Jurgen Brem , Christopher J. Schofield Communications Biology 7 DOI: 10.1038/s42003-024-06785-3 Open Access Show Abstract ▼ Abstract: The essential L,D-transpeptidase of Mycobacterium tuberculosis (LdtMt2) catalyses the formation of 33 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit LdtMt2 have been hampered by lack of knowledge of how it binds its substrate. To address this gap, we optimised the isolation of natural disaccharide tetrapeptide monomers from the Corynebacterium jeikeium bacterial cell wall through overproduction of the peptidoglycan sacculus. The tetrapeptides were used in binding / turnover assays and biophysical studies on LdtMt2. We determined a crystal structure of wild-type LdtMt2 reacted with its natural substrate, the tetrapeptide monomer of the peptidoglycan layer. This structure shows formation of a thioester linking the catalytic cysteine and the donor substrate, reflecting an intermediate in the transpeptidase reaction; it informs on the mode of entrance of the donor substrate into the LdtMt2 active site. The results will be useful in design of LdtMt2 inhibitors, including those based on substrate binding interactions, a strategy successfully employed for other nucleophilic cysteine enzymes.	Sep 2024
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Cell-active small molecule inhibitors validate the SNM1A DNA repair nuclease as a cancer target Marcin Bielinski , Lucy R. Henderson , Yuliana Yosaatmadja , Lonnie P. Swift , Hannah T. Baddock , Matthew J. Bowen , Jurgen Brem , Philip S. Jones , Stuart P. Mcelroy , Angus Morrison , Michael Speake , Stan Van Boeckel , Els Van Doornmalen , Jan Van Groningen , Helma Van Den Hurk , Opher Gileadi , Joseph A Newman , Peter J. Mchugh , Christopher J. Schofield Chemical Science 60 DOI: 10.1039/D4SC00367E Diamond Proposal Number(s): [19301] Open Access Show Abstract ▼ Abstract: The three human SNM1 metallo-β-lactamase fold nucleases (SNM1A–C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site. Structure and turnover assay-guided optimization enabled the identification of potent quinazoline–hydroxamic acid containing inhibitors, which bind in a manner where the hydroxamic acid displaces the hydrolytic water and the quinazoline ring occupies a substrate nucleobase binding site. Cellular assays reveal that SNM1A inhibitors cause sensitisation to, and defects in the resolution of, cisplatin-induced DNA damage, validating the tractability of MBL fold nucleases as cancer drug targets.	Apr 2024
I03-Macromolecular Crystallography	High-throughput screen with the L,D-transpeptidase LdtMt2 of Mycobacterium tuberculosis reveals novel classes of covalently reacting inhibitors Mariska De Munnik , Pauline Lang , Francisco De Dios Anton , Monica Cacho , Robert H. Bates , Jurgen Brem , Beatriz Rodríguez Miquel , Christopher J. Schofield Chemical Science DOI: 10.1039/D2SC06858C Open Access Show Abstract ▼ Abstract: Disruption of bacterial cell wall biosynthesis in Mycobacterium tuberculosis is a promising target for treating tuberculosis. The L,D-transpeptidase LdtMt2, which is responsible for the formation of 3□(→)3 cross-links in the cell wall peptidoglycan, has been identified as essential for M. tuberculosis virulence. We optimised a high-throughput assay for LdtMt2, and screened a targeted library of ~10.000 electrophilic compounds. Potent inhibitor classes were identified, including established (e.g. β-lactams) and unexplored covalently reacting electrophilic groups (e.g., cyanamides). Protein-observed mass spectrometric studies reveal most classes to react covalently and irreversibly with the LdtMt2 catalytic cysteine (Cys354). Crystallographic analyses of seven representative inhibitors reveal induced fit involving a loop enclosing the LdtMt2 active site. Several of the identified compounds have a bactericidal effect on M. tuberculosis within macrophages, one with an MIC50 value of ~1 µM. The results provide leads for the development of new covalently reaction inhibitors of LdtMt2 and other nucleophilic cysteine enzymes.	May 2023
I03-Macromolecular Crystallography	Studies on enmetazobactam clarify mechanisms of widely used β-lactamase inhibitors Pauline A. Lang , Ritu Raj , Anthony Tumber , Christopher T. Lohans , Patrick Rabe , Carol V. Robinson , Jurgen Brem , Christopher J. Schofield Proceedings Of The National Academy Of Sciences 119 DOI: 10.1073/pnas.2117310119 Diamond Proposal Number(s): [19458, 23459] Open Access Show Abstract ▼ Abstract: β-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine β-lactamase (SBL)-catalyzed hydrolysis. The scope of β-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone–derived acyl–enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl–enzyme complexes.	May 2022
I04-1-Macromolecular Crystallography (fixed wavelength)	Studies on the reactions of biapenem with VIM metallo β-lactamases and the serine β-lactamase KPC-2 Anka Lucic , Tika R. Malla , Karina Calvopina , Catherine L. Tooke , Jurgen Brem , Michael A. Mcdonough , James Spencer , Christopher J. Schofield Antibiotics 11 DOI: 10.3390/antibiotics11030396 Diamond Proposal Number(s): [18069] Open Access Show Abstract ▼ Abstract: Carbapenems are important antibacterials and are both substrates and inhibitors of some β-lactamases. We report studies on the reaction of the unusual carbapenem biapenem, with the subclass B1 metallo-β-lactamases VIM-1 and VIM-2 and the class A serine-β-lactamase KPC-2. X-ray diffraction studies with VIM-2 crystals treated with biapenem reveal the opening of the β-lactam ring to form a mixture of the (2S)-imine and enamine complexed at the active site. NMR studies on the reactions of biapenem with VIM-1, VIM-2, and KPC-2 reveal the formation of hydrolysed enamine and (2R)- and (2S)-imine products. The combined results support the proposal that SBL/MBL-mediated carbapenem hydrolysis results in a mixture of tautomerizing enamine and (2R)- and (2S)-imine products, with the thermodynamically favoured (2S)-imine being the major observed species over a relatively long-time scale. The results suggest that prolonging the lifetimes of β-lactamase carbapenem complexes by optimising tautomerisation of the nascently formed enamine to the (2R)-imine and likely more stable (2S)-imine tautomer is of interest in developing improved carbapenems.	Mar 2022
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors Jurgen Brem , Tharindi Panduwawala , Jon Ulf Hansen , Joanne Hewitt , Edgars Liepins , Pawel Donets , Laura Espina , Alistair J. M. Farley , Kirill Shubin , Gonzalo Gomez Campillos , Paula Kiuru , Shifali Shishodia , Daniel Krahn , Robert K. Leśniak , Juliane Schmidt , Karina Calvopina , María-Carmen Turrientes , Madeline E. Kavanagh , Dmitrijs Lubriks , Philip Hinchliffe , Gareth W. Langley , Ali F. Aboklaish , Anders Eneroth , Maria Backlund , Andrei G. Baran , Elisabet I. Nielsen , Michael Speake , Janis Kuka , John Robinson , Solveiga Grinberga , Lindsay Robinson , Michael A. Mcdonough , Anna M. Rydzik , Thomas M. Leissing , Juan Carlos Jimenez-Castellanos , Matthew B. Avison , Solange Da Silva Pinto , Andrew D. Pannifer , Marina Martjuga , Emma Widlake , Martins Priede , Iva Hopkins Navratilova , Marek Gniadkowski , Anna Karin Belfrage , Peter Brandt , Jari Yli-Kauhaluoma , Eric Bacque , Malcolm G. P. Page , Fredrik Björkling , Jonathan M. Tyrrell , James Spencer , Pauline A. Lang , Pawel Baranczewski , Rafael Cantón , Stuart P. Mcelroy , Philip S. Jones , Fernando Baquero , Edgars Suna , Angus Morrison , Timothy R. Walsh , Christopher J. Schofield Nature Chemistry 59 DOI: 10.1038/s41557-021-00831-x Open Access Show Abstract ▼ Abstract: Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-β-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential β-lactamase stable β-lactam mimics. Subsequent structure–activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL–carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.	Dec 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis Patrick Rabe , Jos J. A. G. Kamps , Kyle D. Sutherlin , James D. S. Linyard , Pierre Aller , Cindy C. Pham , Mikako Makita , Ian Clifton , Michael A. Mcdonough , Thomas M. Leissing , Denis Shutin , Pauline A. Lang , Agata Butryn , Jurgen Brem , Sheraz Gul , Franklin D. Fuller , In-Sik Kim , Mun Hon Cheah , Thomas Fransson , Asmit Bhowmick , Iris D. Young , Lee O'Riordan , Aaron S. Brewster , Ilaria Pettinati , Margaret Doyle , Yasumasa Joti , Shigeki Owada , Kensuke Tono , Alexander Batyuk , Mark S. Hunter , Roberto Alonso-Mori , Uwe Bergmann , Robin L. Owen , Nicholas K. Sauter , Timothy D. W. Claridge , Carol V. Robinson , Vittal K. Yachandra , Junko Yano , Jan F. Kern , Allen M. Orville , Christopher J. Schofield Science Advances 7 DOI: 10.1126/sciadv.abh0250 Diamond Proposal Number(s): [23459, 19458] Open Access Show Abstract ▼ Abstract: Isopenicillin N synthase (IPNS) catalyzes the unique reaction of L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.	Aug 2021
I24-Microfocus Macromolecular Crystallography	An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography Agata Butryn , Philipp S. Simon , Pierre Aller , Philip Hinchliffe , Ramzi N. Massad , Gabriel Leen , Catherine L. Tooke , Isabel Bogacz , In-Sik Kim , Asmit Bhowmick , Aaron S. Brewster , Nicholas E. Devenish , Jurgen Brem , Jos J. A. G. Kamps , Pauline A. Lang , Patrick Rabe , Danny Axford , John H. Beale , Bradley Davy , Ali Ebrahim , Julien Orlans , Selina L. S. Storm , Tiankun Zhou , Shigeki Owada , Rie Tanaka , Kensuke Tono , Gwyndaf Evans , Robin L. Owen , Frances A. Houle , Nicholas K. Sauter , Christopher J. Schofield , James Spencer , Vittal K. Yachandra , Junko Yano , Jan F. Kern , Allen M. Orville Nature Communications 12 DOI: 10.1038/s41467-021-24757-7 Diamond Proposal Number(s): [19458, 25260] Open Access Show Abstract ▼ Abstract: Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.	Jul 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	High-throughput crystallography reveals boron-containing inhibitors of a penicillin-binding protein with di- and tricovalent binding modes Hector Newman , Alen Krajnc , Domenico Bellini , Charles J. Eyermann , Grant A. Boyle , Neil Paterson , Katherine E. Mcauley , Robert Lesniak , Mukesh Gangar , Frank Von Delft , Jurgen Brem , Kelly Chibale , Christopher J. Schofield , Christopher G. Dowson Journal Of Medicinal Chemistry 64 DOI: 10.1021/acs.jmedchem.1c00717 Diamond Proposal Number(s): [17884] Open Access Show Abstract ▼ Abstract: The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boron-containing inhibitors are potent serine-β-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) β-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by β-lactamase-driven resistance.	Jul 2021

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