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1,229 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
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	The role of the light chain in the structure and binding activity of two cattle antibodies that neutralize bovine respiratory syncytial virus Jingshan Ren , Joanne E. Nettleship , Gemma Harris , William Mwangi , Nahid Rhaman , Clare Grant , Abhay Kotecha , Elizabeth Fry , Bryan Charleston , David I. Stuart , John Hammond , Raymond J. Owens Molecular Immunology 112, 123 - 130 DOI: 10.1016/j.molimm.2019.04.026 Diamond Proposal Number(s): [10627, 14744] Open Access Show Abstract ▼ Abstract: Cattle antibodies have unusually long CDR3 loops in their heavy chains (HCs), and limited light chain (LC) diversity, raising the question of whether these mask the effect of LC variation on antigen recognition. We have investigated the role of the LC in the structure and activity of two neutralizing cattle antibodies (B4 and B13) that bind the F protein of bovine respiratory syncytial virus (bRSV). Recombinant Fab fragments of B4 and B13 bound bRSV infected cells and showed similar affinities for purified bRSV F protein. Exchanging the LCs between the Fab fragments produced hybrid Fabs: B13* (B13 HC/B4 LC) and B4* (B4 HC/B13 LC). The affinity of B13* to the F protein was found to be two-fold lower than B13 whilst the binding affinity of B4* was reduced at least a hundred-fold compared to B4 such that it no longer bound to bRSV infected cells. Comparison of the structures of B4 and B13 with their LC exchanged counterparts B4* and B13* showed that paratope of the HC variable domain (VH) of B4 was disrupted on pairing with the B13 LC, consistent with the loss of binding activity. By contrast, B13 H3 adopts a similar conformation when paired with either B13 or B4 LCs. These observations confirm the expected key role of the extended H3 loop in antigen-binding by cattle antibodies but also show that the quaternary LC/HC subunit interaction can be crucial for its presentation and thus the LC variable domain (VL) is also important for antigen recognition.	Aug 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Identification and structural analysis of the tripartite α-pore forming toxin of Aeromonas hydrophila Jason S. Wilson , Alicia M. Churchill-angus , Simon P. Davies , Svetlana E. Sedelnikova , Svetomir B. Tzokov , John B. Rafferty , Per A. Bullough , Claudine Bisson , Patrick J. Baker Nature Communications 10, 2900 DOI: 10.1038/s41467-019-10777-x Diamond Proposal Number(s): [8987, 12788, 17773] Open Access Show Abstract ▼ Abstract: The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.	Jul 2019
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Novel and improved crystal structures of Haemophilus influenzae, E. coli and P. aeruginosa Penicillin-Binding-Protein 3 (PBP3) and N. gonorrhoeae PBP2: Towards a better understanding of β-lactam target-mediated resistance Dom Bellini , Lizbe Koekemoer , Hector Newman , Christopher G. Dowson Journal Of Molecular Biology DOI: 10.1016/j.jmb.2019.07.010 Show Abstract ▼ Abstract: Even with the emergence of antibiotic resistance, penicillin and the wider family of beta-lactams have remained the single most important family of antibiotics. The periasmic/extracytoplasmic targets of penicillin are a family of enzymes with a highly conserved catalytic activity involved in the final stage of bacterial cell wall (peptidoglycan, PG) biosynthesis. Named after their ability to bind penicillin, rather than their catalytic activity these key targets are called penicillin-binding proteins (PBPs).Resistance is predominantly mediated by reducing the target drug concentration via beta-lactamases, however, naturally transformable bacteria have also acquired target mediated resistance by inter-species recombination. Here we focus on structural based interpretations of amino acid alterations associated with the emergence of resistance within clinical isolates and include new PBP3 structures along with new, and improved, PBP-beta-lactam co-structures.	Jul 2019
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Discovery of a first-in-class Receptor Interacting Protein 2 (RIP2) kinase specific clinical candidate, 2-((4-(Benzo[ d ]thiazol-5-ylamino)-6-( tert -butylsulfonyl)quinazolin-7-yl)oxy)ethyl dihydrogen phosphate, for the treatment of inflammatory diseases Pamela A. Haile , Linda N. Casillas , Bartholomew J. Votta , Gren Z. Wang , Adam K. Charnley , Xiaoyang Dong , Michael J. Bury , Joseph J. Romano , John F. Mehlmann , Bryan W. King , Karl F. Erhard , Charles R. Hanning , David B. Lipshutz , Biva M. Desai , Carol A. Capriotti , Michelle C. Schaeffer , Scott B. Berger , Mukesh K. Mahajan , Michael A. Reilly , Rakesh Nagilla , Elizabeth J. Rivera , Helen H. Sun , John K. Kenna , Allison M. Beal , Michael T. Ouellette , Mike Kelly , Gillian Stemp , Máire A. Convery , Anna Vossenkämper , Thomas T. Macdonald , Peter J. Gough , John Bertin , Robert W. Marquis Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.9b00575 Diamond Proposal Number(s): [1195, 5799] Show Abstract ▼ Abstract: RIP2 kinase has been identified as a key signal transduction partner in the NOD2 pathway contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP2 kinase or its signaling partners on the NOD2 pathway that are suitable for advancement into the clinic have yet to be described. Herein, we report our discovery and profile of the prodrug clinical compound, inhibitor 3, currently in phase 1 clinical studies. Compound 3 potently binds to RIP2 kinase with good kinase specificity and has excellent activity in blocking many proinflammatory cytokine responses in vivo and in human IBD explant samples. The highly favorable physicochemical and ADMET properties of 3 combined with high potency led to a predicted low oral dose in humans.	Jul 2019
I03-Macromolecular Crystallography	Crystal structures and nuclear magnetic resonance studies of the Apo form of the c-MYC:MAX bHLHZip complex reveal a helical basic region in the absence of DNA Susan Sammak , Najoua Hamdani , Fabrice Gorrec , Mark D. Allen , Stefan M. V. Freund , Mark Bycroft , Giovanna Zinzalla Biochemistry DOI: 10.1021/acs.biochem.9b00296 Show Abstract ▼ Abstract: The c-MYC transcription factor is a master regulator of cell growth and proliferation and is an established target for cancer therapy. This basic helix–loop–helix Zip protein forms a heterodimer with its obligatory partner MAX, which binds to DNA via the basic region. Considerable research efforts are focused on targeting the heterodimerization interface and the interaction of the complex with DNA. The only available crystal structure is that of a c-MYC:MAX complex artificially tethered by an engineered disulfide linker and prebound to DNA. We have carried out a detailed structural analysis of the apo form of the c-MYC:MAX complex, with no artificial linker, both in solution using nuclear magnetic resonance (NMR) spectroscopy and by X-ray crystallography. We have obtained crystal structures in three different crystal forms, with resolutions between 1.35 and 2.2 Å, that show extensive helical structure in the basic region. Determination of the α-helical propensity using NMR chemical shift analysis shows that the basic region of c-MYC and, to a lesser extent, that of MAX populate helical conformations. We have also assigned the NMR spectra of the c-MYC basic helix–loop–helix Zip motif in the absence of MAX and showed that the basic region has an intrinsic helical propensity even in the absence of its dimerization partner. The presence of helical structure in the basic regions in the absence of DNA suggests that the molecular recognition occurs via a conformational selection rather than an induced fit. Our work provides both insight into the mechanism of DNA binding and structural information to aid in the development of MYC inhibitors.	Jul 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors (Part 2) Erik De Heuvel , Abhimanyu K. Singh , Pierre Boronat , Albert J. Kooistra , Tiffany Van Der Meer , Payman Sadek , Antoni R. Blaazer , Nathan C. Shaner , Daphne S. Bindels , Guy Caljon , Louis Maes , Geert Jan Sterk , Marco Siderius , Michael Oberholzer , Iwan J. P. De Esch , David G. Brown , Rob Leurs Bioorganic & Medicinal Chemistry DOI: 10.1016/j.bmc.2019.06.026 Diamond Proposal Number(s): [13689, 16207] Open Access Show Abstract ▼ Abstract: Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action.	Jul 2019
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural basis for recruitment of DAPK1 to the KLHL20 E3 ligase Zhuoyao Chen , Sarah Picaud , Panagis Filippakopoulos , Vincenzo D'angiolella , Alex N. Bullock Structure DOI: 10.1016/j.str.2019.06.005 Diamond Proposal Number(s): [15433] Open Access Show Abstract ▼ Abstract: BTB-Kelch proteins form the largest subfamily of Cullin-RING E3 ligases, yet their substrate complexes are mapped and structurally characterized only for KEAP1 and KLHL3. KLHL20 is a related CUL3-dependent ubiquitin ligase linked to autophagy, cancer, and Alzheimer's disease that promotes the ubiquitination and degradation of substrates including DAPK1, PML, and ULK1. We identified an “LPDLV”-containing motif in the DAPK1 death domain that determines its recruitment and degradation by KLHL20. A 1.1-Å crystal structure of a KLHL20 Kelch domain-DAPK1 peptide complex reveals DAPK1 binding as a loose helical turn that inserts deeply into the central pocket of the Kelch domain to contact all six blades of the β propeller. Here, KLHL20 forms salt-bridge and hydrophobic interactions including tryptophan and cysteine residues ideally positioned for covalent inhibitor development. The structure highlights the diverse binding modes of β-propeller domains versus linear grooves and suggests a new target for structure-based drug design.	Jul 2019
I03-Macromolecular Crystallography	Structure of KAP1 tripartite motif identifies molecular interfaces required for retroelement silencing Guido A. Stoll , Shun-ichiro Oda , Zheng-shan Chong , Minmin Yu , Stephen H. Mclaughlin , Yorgo Modis Proceedings Of The National Academy Of Sciences 60 DOI: 10.1073/pnas.1901318116 Diamond Proposal Number(s): [15916] Open Access Show Abstract ▼ Abstract: Retroviruses can integrate their DNA into the host-cell genome. Inherited retroviral DNA and other transposable elements account for more than half of the human genome. Transposable elements must be tightly regulated to restrict their proliferation and prevent toxic gene expression. KAP1/TRIM28 is an essential regulator of transposable element transcription. We determined the crystal structure of the KAP1 TRIM. The structure identifies a protein–protein interaction site required for recruitment of KAP1 to transposable elements. An epigenetic gene silencing assay confirms the importance of this site for KAP1-dependent silencing. We also show that KAP1 self-assembles in solution, but this self-assembly is not required for silencing. Our work provides insights into KAP1-dependent silencing and tools for expanding our mechanistic understanding of this process.	Jul 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	The hydrolase LpqI primes mycobacterial peptidoglycan recycling Patrick J. Moynihan , Ian T. Cadby , Natacha Veerapen , Monika Jankute , Marialuisa Crosatti , Galina V. Mukamolova , Andrew L. Lovering , Gurdyal S. Besra Nature Communications 10, 2647 DOI: 10.1038/s41467-019-10586-2 Diamond Proposal Number(s): [14692] Open Access Show Abstract ▼ Abstract: Growth and division by most bacteria requires remodelling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides, which are recycled in many model organisms. Bacteria and hosts can harness the unique nature of muropeptides as a signal for cell wall damage and infection, respectively. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core mycobacterial gene lpqI, encodes an authentic NagZ β-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.	Jun 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structure-guided fragment-based drug discovery at the synchrotron: screening binding sites and correlations with hotspot mapping Sherine E. Thomas , Patrick Collins , Rory Hennell James , Vitor Mendes , Sitthivut Charoensutthivarakul , Chris Radoux , Chris Abell , Anthony G. Coyne , R. Andres Floto , Frank Von Delft , Tom Blundell Philosophical Transactions Of The Royal Society A: Mathematical, Physical And Engineering Sciences 377 DOI: 10.1098/rsta.2018.0422 Open Access Show Abstract ▼ Abstract: Structure-guided drug discovery emerged in the 1970s and 1980s, stimulated by the three-dimensional structures of protein targets that became available, mainly through X-ray crystal structure analysis, assisted by the development of synchrotron radiation sources. Structures of known drugs or inhibitors were used to guide the development of leads. The growth of high-throughput screening during the late 1980s and the early 1990s in the pharmaceutical industry of chemical libraries of hundreds of thousands of compounds of molecular weight of approximately 500 Da was impressive but still explored only a tiny fraction of the chemical space of the predicted 1040 drug-like compounds. The use of fragments with molecular weights less than 300 Da in drug discovery not only decreased the chemical space needing exploration but also increased promiscuity in binding targets. Here we discuss advances in X-ray fragment screening and the challenge of identifying sites where fragments not only bind but can be chemically elaborated while retaining their positions and binding modes. We first describe the analysis of fragment binding using conventional X-ray difference Fourier techniques, with Mycobacterium abscessus SAICAR synthetase (PurC) as an example. We observe that all fragments occupy positions predicted by computational hotspot mapping. We compare this with fragment screening at Diamond Synchrotron Light Source XChem facility using PanDDA software, which identifies many more fragment hits, only some of which bind to the predicted hotspots. Many low occupancy sites identified may not support elaboration to give adequate ligand affinity, although they will likely be useful in drug discovery as ‘warm spots’ for guiding elaboration of fragments bound at hotspots. We discuss implications of these observations for fragment screening at the synchrotron sources.	Jun 2019

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