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

Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Targeting ER α-glucosidase I with a single-dose iminosugar treatment protects against lethal influenza and dengue virus infections Kelly Warfield , Dominic S. Alonzi , Johan C. Hill , Alessandro T. Caputo , Pietro Roversi , John I. Kiappes , Nicholas Sheets , Matthew Duchars , Raymond Dwek , Julia Biggins , Dale L. Barnard , Sujan Shresta , Anthony Treston , Nicole Zitzmann Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.0c00067 Show Abstract ▼ Abstract: Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER alpha-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here we show that a single dose of UV-4B (the hydrochloride salt form of N-(9′-methoxynonyl)-1- deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 hours post-infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.	Mar 2020
	Target highlights from the first post-PSI CASP experiment (CASP12, May-August 2016) Andriy Kryshtafovych , Reinhard Albrecht , Arnaud Basle , Pedro Bule , Alessandro T. Caputo , Ana Luisa Carvalho , Kinlin L. Chao , Ron Diskin , Krzysztof Fidelis , Carlos M. G. A. Fontes , Folmer Fredslund , Harry J. Gilbert , Celia W. Goulding , Marcus D. Hartmann , Christopher S. Hayes , Osnat Herzberg , Johan C. Hill , Andrzej Joachimiak , Gert-wieland Kohring , Roman I. Koning , Leila Lo Leggio , Marco Mangiagalli , Karolina Michalska , John Moult , Shabir Najmudin , Marco Nardini , Valentina Nardone , Didier Ndeh , Thanh-hong Nguyen , Guido Pintacuda , Sandra Postel , Mark J. Van Raaij , Pietro Roversi , Amir Shimon , Abhimanyu K. Singh , Eric J. Sundberg , Kaspars Tars , Nicole Zitzmann , Torsten Schwede Proteins: Structure, Function, And Bioinformatics 86, 27 - 50 DOI: 10.1002/prot.25392 Show Abstract ▼ Abstract: The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.	Mar 2018
I04-1-Macromolecular Crystallography (fixed wavelength) Krios I-Titan Krios I at Diamond	Interdomain conformational flexibility underpins the activity of UGGT, the eukaryotic glycoprotein secretion checkpoint Pietro Roversi , Lucia Marti , Alessandro T. Caputo , Dominic S. Alonzi , Johan C. Hill , Kyle C. Dent , Abhinav Kumar , Mikail D. Levasseur , Andrea Lia , Thomas Waksman , Souradeep Basu , Yentli Soto Albrecht , Kristin Qian , James Patrick Mcivor , Colette B. Lipp , Dritan Siliqi , Snezana Vasilijevic , Shabaz Mohammed , Petra Lukacik , Martin A. Walsh , Angelo Santino , Nicole Zitzmann Proceedings Of The National Academy Of Sciences Pnas DOI: 10.1073/pnas.1703682114 Show Abstract ▼ Abstract: Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.	Jun 2017
I02-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme Jiangyu Yan , Thomas R. Beattie , Adriana L. Rojas , Kelly Schermerhorn , Tamzin Gristwood , Jonathan C. Trinidad , Sonja V. Albers , Pietro Roversi , Andrew F. Gardner , Nicola G. A. Abrescia , Stephen D. Bell Nature Communications 8 DOI: 10.1038/ncomms15075 Diamond Proposal Number(s): [9063, 10130] Open Access Show Abstract ▼ Abstract: Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.	May 2017
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals Alessandro T. Caputo , Dominic S. Alonzi , Lucia Marti , Ida-barbara Reca , John Kiappes , Weston B. Struwe , Alice Cross , Souradeep Basu , Ed Lowe , Benoit Darlot , Angelo Santino , Pietro Roversi , Nicole Zitzmann Proceedings Of The National Academy Of Sciences 113, E4630 - E4638 DOI: 10.1073/pnas.1604463113 Diamond Proposal Number(s): [9306] Show Abstract ▼ Abstract: The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme’s quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme’s activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.	Aug 2016
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Super-complexes of adhesion GPCRs and neural guidance receptors Verity A. Jackson , Shahid Mehmood , Matthieu Chavent , Pietro Roversi , Maria Carrasquero , Daniel Del Toro , Goenuel Seyit-bremer , Fanomezana M. Ranaivoson , Davide Comoletti , Mark S. P. Sansom , Carol V. Robinson , Rüdiger Klein , Elena Seiradake Nature Communications 7 DOI: 10.1038/ncomms11184 Diamond Proposal Number(s): [9306, 8423, 1747] Open Access Show Abstract ▼ Abstract: Latrophilin adhesion-GPCRs (Lphn1–3 or ADGRL1–3) and Unc5 cell guidance receptors (Unc5A–D) interact with FLRT proteins (FLRT1–3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger ‘super-complex’ (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.	Apr 2016
I04-Macromolecular Crystallography	Structural Basis of Latrophilin-FLRT Interaction Verity Jackson , Daniel Del toro , Maria Carrasquero , Pietro Roversi , Karl Harlos , Rüdiger Klein , Elena Seiradake Structure DOI: 10.1016/j.str.2015.01.013 PMID: 25728924 Diamond Proposal Number(s): [10627] Open Access Show Abstract ▼ Abstract: Jackson et al. describe a crystal structure of mLPHN3 lectin and olfactomedin-like (Olf) domains, revealing the Olf b-propeller fold and calcium-binding site. Assays using HeLa cells and cortical neurons reveal a bi-functional role for Olf and its ligand FLRT, leading to HeLa cell adhesion and neuron repulsion.	Apr 2015
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding Paul Chappell , El Kahina Meziane , Michael Harrison , Łukasz Magiera , Clemens Hermann , Laura Mears , Antoni G Wrobel , Charlotte Durant , Lise Lotte Nielsen , Søren Buus , Nicola Ternette , William Mwangi , Colin Butter , Venugopal Nair , Trudy Ahyee , Richard Duggleby , Alejandro Madrigal , Pietro Roversi , Susan Lea , Jim Kaufman Elife 4 DOI: 10.7554/eLife.05345 PMID: 25860507 Diamond Proposal Number(s): [9306] Open Access Show Abstract ▼ Abstract: Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. - See more at: http://elifesciences.org/content/4/e05345#sthash.zKFHdAeD.dpuf	Apr 2015
I03-Macromolecular Crystallography	A complex iron-calcium cofactor catalyzing phosphotransfer chemistry S. C. Yong , P. Roversi , J. Lillington , F. Rodriguez , M. Krehenbrink , O. B. Zeldin , E. F. Garman , S. M. Lea , B. C. Berks Science 345, 1170 - 1173 Science Magazine DOI: 10.1126/science.1254237 PMID: 25190793 Diamond Proposal Number(s): [7495, 9306] Open Access Show Abstract ▼ Abstract: Alkaline phosphatases play a crucial role in phosphate acquisition by microorganisms. To expand our understanding of catalysis by this class of enzymes, we have determined the structure of the widely occurring microbial alkaline phosphatase PhoX. The enzyme contains a complex active-site cofactor comprising two antiferromagnetically coupled ferric iron ions (Fe(3+)), three calcium ions (Ca(2+)), and an oxo group bridging three of the metal ions. Notably, the main part of the cofactor resembles synthetic oxide-centered triangular metal complexes. Structures of PhoX-ligand complexes reveal how the active-site metal ions bind substrate and implicate the cofactor oxo group in the catalytic mechanism. The presence of iron in PhoX raises the possibility that iron bioavailability limits microbial phosphate acquisition.	Sep 2014
I04-1-Macromolecular Crystallography (fixed wavelength)	Identification and characterization of the missing terminal enzyme for siroheme biosynthesis in [alpha]-proteobacteria Shilpa Bali , Sarah Rollauer , Pietro Roversi , Evelyne Raux-deery , Susan Lea , Martin J. Warren , Stuart J. Ferguson Molecular Microbiology 92 (1), 153 - 163 DOI: 10.1111/mmi.12542 PMID: 24673795 Diamond Proposal Number(s): [9306] Open Access Show Abstract ▼ Abstract: It has recently been shown that the biosynthetic route for both the d1-haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1-haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorinferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorinferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis.	Apr 2014

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