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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
I03-Macromolecular Crystallography Krios II-Titan Krios II at Diamond	Structural insights into Frizzled3 through nanobody modulators James Hillier , Yuguang Zhao , Loic Carrique , Tomas Malinauskas , Reinis R. Ruza , Tao-Hsin Chang , Gangshun Yi , Helen M. E. Duyvesteyn , Jing Yu , Weixian Lu , Els Pardon , Jan Steyaert , Yanan Zhu , Tao Ni , E. Yvonne Jones Nature Communications 15 DOI: 10.1038/s41467-024-51451-1 Diamond Proposal Number(s): [19946, 28713] Open Access Show Abstract ▼ Abstract: The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating β-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.	Aug 2024
I03-Macromolecular Crystallography I23-Long wavelength MX I24-Microfocus Macromolecular Crystallography	Molecular mechanism of BMP signal control by Twisted gastrulation Tomas Malinauskas , Gareth Moore , Amalie F. Rudolf , Holly Eggington , Hayley L. Belnoue-Davis , Kamel El Omari , Samuel C. Griffiths , Rachel E. Woolley , Ramona Duman , Armin Wagner , Simon J. Leedham , Clair Baldock , Hilary L. Ashe , Christian Siebold Nature Communications 15 DOI: 10.1038/s41467-024-49065-8 Diamond Proposal Number(s): [28534, 14744, 19946] Open Access Show Abstract ▼ Abstract: Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.	Jun 2024
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	GluD1 binds GABA and controls inhibitory plasticity Laura Piot , Christina Heroven , Simon Bossi , Joseph Zamith , Tomas Malinauskas , Chris Johnson , Doris Wennagel , David Stroebel , Cécile Charrier , A. Radu Aricescu , Laetitia Mony , Pierre Paoletti Science DOI: 10.1126/science.adf3406 Show Abstract ▼ Abstract: Fast synaptic neurotransmission in the vertebrate central nervous system relies primarily on ionotropic glutamate receptors (iGluRs), that drive neuronal excitation, and type A γ-aminobutyric acid receptors (GABAARs), responsible for neuronal inhibition. However, the GluD1 receptor, an iGluR family member, is present at both excitatory and inhibitory synapses. Whether and how GluD1 activation may impact inhibitory neurotransmission is unknown. Here, using a combination of biochemical, structural and functional analyses, we demonstrate that GluD1 binds GABA, an unprecedented feature for iGluRs. GluD1 activation produces long-lasting enhancement of GABAergic synaptic currents in the adult mouse hippocampus through a non-ionotropic mechanism dependent on trans-synaptic anchoring. The identification of GluD1 as a GABA receptor that controls inhibitory synaptic plasticity challenges the classical dichotomy between glutamatergic and GABAergic receptors.	Dec 2023
I04-Macromolecular Crystallography	The molecular basis of drug selectivity for α5 subunit-containing GABAA receptors Vikram Babu Kasaragod , Tomas Malinauskas , Ayla A. Wahid , Judith Lengyel , Frederic Knoflach , Steven W. Hardwick , Charlotte F. Jones , Wan-Na Chen , Xavier Lucas , Kamel El Omari , Dimitri Y. Chirgadze , A. Radu Aricescu , Giuseppe Cecere , Maria-Clemencia Hernandez , Paul S. Miller Nature Structural & Molecular Biology 90 DOI: 10.1038/s41594-023-01133-1 Diamond Proposal Number(s): [10627] Open Access Show Abstract ▼ Abstract: α5 subunit-containing γ-aminobutyric acid type A (GABAA) receptors represent a promising drug target for neurological and neuropsychiatric disorders. Altered expression and function contributes to neurodevelopmental disorders such as Dup15q and Angelman syndromes, developmental epilepsy and autism. Effective drug action without side effects is dependent on both α5-subtype selectivity and the strength of the positive or negative allosteric modulation (PAM or NAM). Here we solve structures of drugs bound to the α5 subunit. These define the molecular basis of binding and α5 selectivity of the β-carboline, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), type II benzodiazepine NAMs, and a series of isoxazole NAMs and PAMs. For the isoxazole series, each molecule appears as an ‘upper’ and ‘lower’ moiety in the pocket. Structural data and radioligand binding data reveal a positional displacement of the upper moiety containing the isoxazole between the NAMs and PAMs. Using a hybrid molecule we directly measure the functional contribution of the upper moiety to NAM versus PAM activity. Overall, these structures provide a framework by which to understand distinct modulator binding modes and their basis of α5-subtype selectivity, appreciate structure–activity relationships, and empower future structure-based drug design campaigns.	Oct 2023
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Hedgehog-Interacting Protein is a multimodal antagonist of Hedgehog signalling Samuel C. Griffiths , Rebekka A. Schwab , Kamel El Omari , Benjamin Bishop , Ellen J. Iverson , Tomas Malinauskas , Ramin Dubey , Mingxing Qian , Douglas F. Covey , Robert J. C. Gilbert , Rajat Rohatgi , Christian Siebold Nature Communications 12 DOI: 10.1038/s41467-021-27475-2 Diamond Proposal Number(s): [19946, 14744] Open Access Show Abstract ▼ Abstract: Hedgehog (HH) morphogen signalling, crucial for cell growth and tissue patterning in animals, is initiated by the binding of dually lipidated HH ligands to cell surface receptors. Hedgehog-Interacting Protein (HHIP), the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling, binds directly to SHH with high nanomolar affinity, sequestering SHH. Here, we report the structure of the HHIP N-terminal domain (HHIP-N) in complex with a glycosaminoglycan (GAG). HHIP-N displays a unique bipartite fold with a GAG-binding domain alongside a Cysteine Rich Domain (CRD). We show that HHIP-N is required to convey full HHIP inhibitory function, likely by interacting with the cholesterol moiety covalently linked to HH ligands, thereby preventing this SHH-attached cholesterol from binding to the HH receptor Patched (PTCH1). We also present the structure of the HHIP C-terminal domain in complex with the GAG heparin. Heparin can bind to both HHIP-N and HHIP-C, thereby inducing clustering at the cell surface and generating a high-avidity platform for SHH sequestration and inhibition. Our data suggest a multimodal mechanism, in which HHIP can bind two specific sites on the SHH morphogen, alongside multiple GAG interactions, to inhibit SHH signalling.	Dec 2021
	Site-specific steric control of SARS-COV-2 spike glycosylation Joel D. Allen , Himanshi Chawla , Firdaus Samsudin , Lorena Zuzic , Aishwary Tukaram Shivgan , Yasunori Watanabe , Wan-Ting He , Sean Callaghan , Ge Song , Peter Yong , Philip J. M. Brouwer , Yutong Song , Yongfei Cai , Helen M. E. Duyvesteyn , Tomas Malinauskas , Joeri Kint , Paco Pino , Maria J. Wurm , Martin Frank , Bing Chen , David I. Stuart , Rogier W. Sanders , Raiees Andrabi , Dennis R. Burton , Sai Li , Peter J. Bond , Max Crispin Biochemistry DOI: 10.1021/acs.biochem.1c00279 Open Access Show Abstract ▼ Abstract: A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.	Jul 2021
B21-High Throughput SAXS I03-Macromolecular Crystallography Krios IV-Titan Krios IV at Diamond	Simultaneous binding of guidance cues NET1 and RGM blocks extracellular NEO1 signaling Ross A. Robinson , Samuel C. Griffiths , Lieke L. Van De Haar , Tomas Malinauskas , Eljo Y. Van Battum , Pavol Zelina , Rebekka A. Schwab , Dimple Karia , Lina Malinauskaite , Sara Brignani , Marleen H. Van Den Munkhof , Özge Düdükcü , Anna A. De Ruiter , Dianne M.a. Van Den Heuvel , Benjamin Bishop , Jonathan Elegheert , A. Radu Aricescu , R. Jeroen Pasterkamp , Christian Siebold Cell 74 DOI: 10.1016/j.cell.2021.02.045 Diamond Proposal Number(s): [19946, 20223] Open Access Show Abstract ▼ Abstract: During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a “trimer-of-trimers” super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.	Mar 2021
	A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses 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 , 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 Nature Communications 12 DOI: 10.1038/s41467-020-20654-7 Open Access Show Abstract ▼ 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.	Jan 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Glypicans shield the Wnt lipid moiety to enable signalling at a distance Ian J. Mcgough , Luca Vecchia , Benjamin Bishop , Tomas Malinauskas , Karen Beckett , Dhira Joshi , Nicola O’reilly , Christian Siebold , E. Yvonne Jones , Jean-Paul Vincent Nature 423 DOI: 10.1038/s41586-020-2498-z Diamond Proposal Number(s): [19946] Show Abstract ▼ Abstract: A relatively small number of proteins have been suggested to act as morphogens—signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1,2,3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4,5,6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.	Jul 2020
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient Daming Zhou , Helen M. E. Duyvesteyn , Cheng-Pin Chen , Chung-Guei Huang , Ting-Hua Chen , Shin-Ru Shih , Yi-Chun Lin , Chien-Yu Cheng , Shu-Hsing Cheng , Yhu-Chering Huang , Tzou-Yien Lin , Che Ma , Jiandong Huo , Loic Carrique , Tomas Malinauskas , Reinis R. Ruza , Pranav Shah , Tiong Kit Tan , Pramila Rijal , Robert F. Donat , Kerry Godwin , Karen R. Buttigieg , Julia A. Tree , Julika Radecke , Neil Paterson , Piyada Supasa , Juthathip Mongkolsapaya , Gavin R. Screaton , Miles W. Carroll , Javier Gilbert-Jaramillo , Michael L. Knight , William James , Raymond J. Owens , James H. Naismith , Alain R. Townsend , Elizabeth E. Fry , Yuguang Zhao , Jingshan Ren , David I. Stuart , Kuan-Ying A. Huang Nature Structural & Molecular Biology 20 DOI: 10.1038/s41594-020-0480-y Diamond Proposal Number(s): [19946, 26983] Show Abstract ▼ Abstract: The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD–EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.	Jul 2020

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