I03-Macromolecular Crystallography
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Joseph A
Newman
,
Angeline E.
Gavard
,
Simone
Lieb
,
Madhwesh C.
Ravichandran
,
Katja
Hauer
,
Patrick
Werni
,
Leonhard
Geist
,
Jark
Böttcher
,
John R
Engen
,
Klaus
Rumpel
,
Matthias
Samwer
,
Mark
Petronczki
,
Opher
Gileadi
Diamond Proposal Number(s):
[19301]
Open Access
Abstract: Loss of WRN, a DNA repair helicase, was identified as a strong vulnerability of microsatellite instable (MSI) cancers, making WRN a promising drug target. We show that ATP binding and hydrolysis are required for genome integrity and viability of MSI cancer cells. We report a 2.2-Å crystal structure of the WRN helicase core (517–1,093), comprising the two helicase subdomains and winged helix domain but not the HRDC domain or nuclease domains. The structure highlights unusual features. First, an atypical mode of nucleotide binding that results in unusual relative positioning of the two helicase subdomains. Second, an additional β-hairpin in the second helicase subdomain and an unusual helical hairpin in the Zn2+ binding domain. Modelling of the WRN helicase in complex with DNA suggests roles for these features in the binding of alternative DNA structures. NMR analysis shows a weak interaction between the HRDC domain and the helicase core, indicating a possible biological role for this association. Together, this study will facilitate the structure-based development of inhibitors against WRN helicase.
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Nov 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Wout
Oosterheert
,
Katerina T.
Xenaki
,
Viviana
Neviani
,
Wouter
Pos
,
Sofia
Doulkeridou
,
Jip
Manshande
,
Nicholas M.
Pearce
,
Loes M. J.
Kroon-batenburg
,
Martin
Lutz
,
Paul M. P.
Van Bergen En Henegouwen
,
Piet
Gros
Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9–EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9–EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9–EWI-F with potential CD9 homo-dimerization at either end provides a “concatenation model” for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.
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Sep 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Petra
Van Der Lelij
,
Joseph A.
Newman
,
Simone
Lieb
,
Julian
Jude
,
Vittorio
Katis
,
Thomas
Hoffmann
,
Matthias
Hinterndorfer
,
Gerd
Bader
,
Norbert
Kraut
,
Mark A.
Pearson
,
Jan-michael
Peters
,
Johannes
Zuber
,
Opher
Gileadi
,
Mark
Petronczki
Diamond Proposal Number(s):
[19301, 18145]
Open Access
Abstract: The cohesin subunit STAG2 has emerged as a recurrently inactivated tumor suppressor in human cancers. Using candidate approaches, recent studies have revealed a synthetic lethal interaction between STAG2 and its paralog STAG1. To systematically probe genetic vulnerabilities in the absence of STAG2, we have performed genome-wide CRISPR screens in isogenic cell lines and identified STAG1 as the most prominent and selective dependency of STAG2-deficient cells. Using an inducible degron system, we show that chemical genetic degradation of STAG1 protein results in the loss of sister chromatid cohesion and rapid cell death in STAG2-deficient cells, while sparing STAG2–wild-type cells. Biochemical assays and X-ray crystallography identify STAG1 regions that interact with the RAD21 subunit of the cohesin complex. STAG1 mutations that abrogate this interaction selectively compromise the viability of STAG2-deficient cells. Our work highlights the degradation of STAG1 and inhibition of its interaction with RAD21 as promising therapeutic strategies. These findings lay the groundwork for the development of STAG1-directed small molecules to exploit synthetic lethality in STAG2-mutated tumors.
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May 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Haiyang
Wu
,
Osmond
Rebello
,
Emmanuelle H.
Crost
,
C. David
Owen
,
Samuel
Walpole
,
Chloe
Bennati-granier
,
Didier
Ndeh
,
Serena
Monaco
,
Thomas
Hicks
,
Anna
Colvile
,
Paulina A.
Urbanowicz
,
Martin A.
Walsh
,
Jesus
Angulo
,
Daniel I. R.
Spencer
,
Nathalie
Juge
Open Access
Abstract: The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1–3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3′-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.
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Apr 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: The scavenger receptor cysteine-rich (SRCR) family of proteins comprises more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ∼110 amino-acid SRCR domain, this class of proteins have widespread functions as antimicrobial molecules, scavenger receptors, and signalling receptors. Despite the high level of structural conservation of SRCR domains, no unifying mechanism for ligand interaction has been described. The SRCR protein SALSA, also known as DMBT1/gp340, is a key player in mucosal immunology. Based on detailed structural data of SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand-binding mechanism for SALSA ligands. The binding interface incorporates a dual cation-binding site, which is highly conserved across the SRCR superfamily. Along with the well-described cation dependency on most SRCR domain–ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for the SRCR class of proteins.
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Feb 2020
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Open Access
Abstract: The emergent zoonotic henipaviruses, Hendra, and Nipah are responsible for frequent and fatal disease outbreaks in domestic animals and humans. Specificity of henipavirus attachment glycoproteins (G) for highly species-conserved ephrin ligands underpins their broad host range and is associated with systemic and neurological disease pathologies. Here, we demonstrate that Cedar virus (CedV)—a related henipavirus that is ostensibly nonpathogenic—possesses an idiosyncratic entry receptor repertoire that includes the common henipaviral receptor, ephrin-B2, but, distinct from pathogenic henipaviruses, does not include ephrin-B3. Uniquely among known henipaviruses, CedV can use ephrin-B1 for cellular entry. Structural analyses of CedV-G reveal a key region of molecular specificity that directs ephrin-B1 utilization, while preserving a universal mode of ephrin-B2 recognition. The structural and functional insights presented uncover diversity within the known henipavirus receptor repertoire and suggest that only modest structural changes may be required to modulate receptor specificities within this group of lethal human pathogens.
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Dec 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9826]
Open Access
Abstract: Centromeric chromatin in fission yeast is distinguished by the presence of nucleosomes containing the histone H3 variant Cnp1CENP-A. Cell cycle–specific deposition of Cnp1 requires the Mis16–Mis18–Mis19 complex, which is thought to direct recruitment of Scm3-chaperoned Cnp1/histone H4 dimers to DNA. Here, we present the structure of the essential Mis18 partner protein Mis19 and describe its interaction with Mis16, revealing a bipartite-binding site. We provide data on the stoichiometry and overall architecture of the complex and provide detailed insights into the Mis18–Mis19 interface.
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Aug 2019
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[14946]
Open Access
Abstract: Adsorption of prebiotic building blocks is proposed to have played a role in the emergence of life on Earth. The experimental and theoretical study of this phenomenon should be guided by our knowledge of the geochemistry of the habitable early Earth environments, which could have spanned a large range of settings. Adsorption being an interfacial phenomenon, experiments can be built around the minerals that probably exhibited the largest specific surface areas and were the most abundant, i.e., phyllosilicates. Our current work aims at understanding how nucleotides, the building blocks of RNA and DNA, might have interacted with phyllosilicates under various physico-chemical conditions. We carried out and refined batch adsorption studies to explore parameters such as temperature, pH, salinity, etc. We built a comprehensive, generalized model of the adsorption mechanisms of nucleotides onto phyllosilicate particles, mainly governed by phosphate reactivity. More recently, we used surface chemistry and geochemistry techniques, such as vibrational spectroscopy, low pressure gas adsorption, X-ray microscopy, and theoretical simulations, in order to acquire direct data on the adsorption configurations and localization of nucleotides on mineral surfaces. Although some of these techniques proved to be challenging, questioning our ability to easily detect biosignatures, they confirmed and complemented our pre-established model.
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Nov 2018
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Open Access
Abstract: Cryo-soft X-ray tomography is an imaging technique that addresses the need for mesoscale imaging of cellular ultrastructure of relatively thick samples without the need for staining or chemical modification. It allows the imaging of cellular ultrastructure to a resolution of 25–40 nm and can be used in correlation with other imaging modalities, such as electron tomography and fluorescence microscopy, to further enhance the information content derived from biological samples. An overview of the technique, discussion of sample suitability and information about sample preparation, data collection and data analysis is presented here. Recent developments and future outlook are also discussed.
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Mar 2018
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I03-Macromolecular Crystallography
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Stephen A.
St-gallay
,
Neil
Bennett
,
Susan E.
Critchlow
,
Nicola
Curtis
,
Gareth
Davies
,
Judit
Debreczeni
,
Nicola
Evans
,
Ian
Hardern
,
Geoff
Holdgate
,
Neil P.
Jones
,
Lindsey
Leach
,
Sarita
Maman
,
Sheila
Mcloughlin
,
Marian
Preston
,
Laurent
Rigoreau
,
Andrew
Thomas
,
Andrew P.
Turnbull
,
Graeme
Walker
,
Jarrod
Walsh
,
Richard A.
Ward
,
Ed
Wheatley
,
Jon
Winter-holt
Diamond Proposal Number(s):
[5735]
Abstract: A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.
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Sep 2017
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