I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26998, 22717, 28172]
Open Access
Abstract: NSP14 is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both activities are essential for the viral life cycle and may be targeted for anti-viral therapeutics. NSP14 forms a complex with NSP10, and this interaction enhances the nuclease but not the methyltransferase activity. We have determined the structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 Å resolution. Comparisons with NSP14/NSP10 complexes reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. We have determined the structure of NSP14 in complex with cap analogue 7MeGpppG, and observe conformational changes within a SAM/SAH interacting loop that plays a key role in viral mRNA capping offering new insights into MTase activity. We perform an X-ray fragment screen on NSP14, revealing 72 hits bound to sites of inhibition in the ExoN and MTase domains. These fragments serve as excellent starting point tools for structure guided development of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.
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Jan 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Joseph A.
Newman
,
Alice
Douangamath
,
Setayesh
Yadzani
,
Yuliana
Yosaatmadja
,
Anthony
Aimon
,
Jose
Brandao-Neto
,
Louise
Dunnett
,
Tyler
Gorrie-Stone
,
Rachael
Skyner
,
Daren
Fearon
,
Matthieu
Schapira
,
Frank
Von Delft
,
Opher
Gileadi
Diamond Proposal Number(s):
[26998]
Open Access
Abstract: There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two “druggable” pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.
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Aug 2021
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15433]
Open Access
Abstract: The SNM1 nucleases which help maintain genome integrity are members of the metallo-β-lactamase (MBL) structural superfamily. Their conserved MBL-β-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5′ phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.
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Aug 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19301]
Open Access
Abstract: Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its β-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.
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Aug 2021
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Nathan David
Wright
,
Patrick
Collins
,
Lizbe
Koekemoer
,
Tobias
Krojer
,
Romain
Talon
,
Elliot
Nelson
,
Mingda
Ye
,
Radoslaw
Nowak
,
Joseph
Newman
,
Jia Tsing
Ng
,
Nick
Mitrovic
,
Helton
Wiggers
,
Frank
Von Delft
Open Access
Abstract: Despite the tremendous success of X-ray cryo-crystallography in recent decades, the transfer of crystals from the drops in which they are grown to diffractometer sample mounts remains a manual process in almost all laboratories. Here, the Shifter, a motorized, interactive microscope stage that transforms the entire crystal-mounting workflow from a rate-limiting manual activity to a controllable, high-throughput semi-automated process, is described. By combining the visual acuity and fine motor skills of humans with targeted hardware and software automation, it was possible to transform the speed and robustness of crystal mounting. Control software, triggered by the operator, manoeuvres crystallization plates beneath a clear protective cover, allowing the complete removal of film seals and thereby eliminating the tedium of repetitive seal cutting. The software, either upon request or working from an imported list, controls motors to position crystal drops under a hole in the cover for human mounting at a microscope. The software automatically captures experimental annotations for uploading to the user's data repository, removing the need for manual documentation. The Shifter facilitates mounting rates of 100–240 crystals per hour in a more controlled process than manual mounting, which greatly extends the lifetime of the drops and thus allows a dramatic increase in the number of crystals retrievable from any given drop without loss of X-ray diffraction quality. In 2015, the first in a series of three Shifter devices was deployed as part of the XChem fragment-screening facility at Diamond Light Source, where they have since facilitated the mounting of over 120 000 crystals. The Shifter was engineered to have a simple design, providing a device that could be readily commercialized and widely adopted owing to its low cost. The versatile hardware design allows use beyond fragment screening and protein crystallography.
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Jan 2021
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Tiong Kit
Tan
,
Pramila
Rijal
,
Rolle
Rahikainen
,
Anthony H.
Keeble
,
Lisa
Schimanski
,
Saira
Hussain
,
Ruth
Harvey
,
Jack W. P.
Hayes
,
Jane C.
Edwards
,
Rebecca K.
Mclean
,
Veronica
Martini
,
Miriam
Pedrera
,
Nazia
Thakur
,
Carina
Conceicao
,
Isabelle
Dietrich
,
Holly
Shelton
,
Anna
Ludi
,
Ginette
Wilsden
,
Clare
Browning
,
Adrian K.
Zagrajek
,
Dagmara
Bialy
,
Sushant
Bhat
,
Phoebe
Stevenson-Leggett
,
Philippa
Hollinghurst
,
Matthew
Tully
,
Katy
Moffat
,
Chris
Chiu
,
Ryan
Waters
,
Ashley
Gray
,
Mehreen
Azhar
,
Valerie
Mioulet
,
Joseph
Newman
,
Amin S.
Asfor
,
Alison
Burman
,
Sylvia
Crossley
,
John A.
Hammond
,
Elma
Tchilian
,
Bryan
Charleston
,
Dalan
Bailey
,
Tobias J.
Tuthill
,
Simon P.
Graham
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Helen M. E.
Duyvesteyn
,
Tomas
Malinauskas
,
Jiandong
Huo
,
Julia A.
Tree
,
Karen R.
Buttigieg
,
Raymond J.
Owens
,
Miles W.
Carroll
,
Rodney S.
Daniels
,
John W.
Mccauley
,
David I.
Stuart
,
Kuan-Ying A.
Huang
,
Mark
Howarth
,
Alain R.
Townsend
Open Access
Abstract: There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.
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Jan 2021
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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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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
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Diamond Proposal Number(s):
[15433]
Abstract: Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors, and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the “nude” phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC), that is different from the more widely-recognized forkhead (FKH) sequence RYAAAYA (R = purine, Y = pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5’-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.
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Dec 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[19301, 15433]
Open Access
Abstract: Ubiquitylation, the posttranslational linkage of ubiquitin moieties to lysines in target proteins, helps regulate a myriad of biological processes. Ubiquitin, and sometimes ubiquitin-homology domains, are recognized by ubiquitin-binding domains, including CUE domains. CUE domains are thus generally thought to function by mediating interactions with ubiquitylated proteins. The chromatin remodeler, SMARCAD1, interacts with KAP1, a transcriptional corepressor. The SMARCAD1-KAP1 interaction is direct and involves the first SMARCAD1 CUE domain (CUE1) and the RBCC domain of KAP1. Here, we present a structural model of the KAP1 RBCC-SMARCAD1 CUE1 complex based on X-ray crystallography. Remarkably, CUE1, a canonical CUE domain, recognizes a cluster of exposed hydrophobic and surrounding charged/amphipathic residues on KAP1, which are presented in the context of a coiled-coil domain, not in a structure resembling ubiquitin. Together, these data suggest that CUE domains may have a wider function than simply recognizing ubiquitin and the ubiquitin-fold.
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Jun 2019
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