Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[25127]
Open Access
Abstract: CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.
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Feb 2023
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I02-Macromolecular Crystallography
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Mostafa
Jamshidiha
,
Thomas
Lanyon-Hogg
,
Charlotte L.
Sutherell
,
Gregory B.
Craven
,
Montse
Tersa
,
Elena
De Vita
,
Delia
Brustur
,
Inmaculada
Perez-Dorado
,
Sarah
Hassan
,
Rita
Petracca
,
Rhodri M.
Morgan
,
Máximo
Sanz-Hernández
,
Jim C.
Norman
,
Alan
Armstrong
,
David J.
Mann
,
Ernesto
Cota
,
Edward W.
Tate
Diamond Proposal Number(s):
[17221, 23620]
Open Access
Abstract: Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein–protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket (‘WF-binding pocket’) via a conserved tryptophan–phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.
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Dec 2021
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Krios I-Titan Krios I at Diamond
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Claire E.
Coupland
,
Sebastian A.
Andrei
,
T. Bertie
Ansell
,
Loic
Carrique
,
Pramod
Kumar
,
Lea
Sefer
,
Rebekka A.
Schwab
,
Eamon F. X.
Byrne
,
Els
Pardon
,
Jan
Steyaert
,
Anthony I.
Magee
,
Thomas
Lanyon-Hogg
,
Mark S. P.
Sansom
,
Edward W.
Tate
,
Christian
Siebold
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
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Dec 2021
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Andrew S.
Bell
,
Zhiyong
Yu
,
Jennie A.
Hutton
,
Megan H.
Wright
,
James A.
Brannigan
,
Daniel
Paape
,
Shirley M.
Roberts
,
Charlotte L.
Sutherell
,
Markus
Ritzefeld
,
Anthony
Wilkinson
,
Deborah F
Smith
,
Robin
Leatherbarrow
,
Edward W.
Tate
Diamond Proposal Number(s):
[1221, 7864]
Open Access
Abstract: The leishmaniases, caused by Leishmania species of protozoan parasites, are neglected tropical diseases with 12-15 million cases worldwide. Current therapeutic approaches are limited by toxicity, resistance and cost. N-Myristoyltransferase (NMT), an enzyme ubiquitous and essential in all eukaryotes, has been validated via genetic and pharmacological methods as a promising antileishmanial target. Here we describe a comprehensive structure activity relationship study of a thienopyrimidine series previously identified in a high throughput screen against Leishmania NMT, across 68 compounds in enzyme- and cell-based assay formats. Using a chemical tagging target engagement biomarker assay we identify the first inhibitor in this series with on-target NMT activity in leishmania parasites. Furthermore, crystal structure analyses of 12 derivatives in complex with Leishmania major NMT revealed key factors important for future structure-guided optimization delivering IMP-105 (43), a compound with modest activity against L. donovani intracellular amastigotes and excellent selectivity (>660-fold) for Leishmania NMT over human NMTs.
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Jun 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[13775]
Abstract: The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5′-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.
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Mar 2020
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I03-Macromolecular Crystallography
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Cyril
Dian
,
Inmaculada
Perez-Dorado
,
Frédéric
Rivière
,
Thomas
Asensio
,
Pierre
Legrand
,
Markus
Ritzefeld
,
Mengjie
Shen
,
Ernesto
Cota
,
Thierry
Meinnel
,
Edward
Tate
,
Carmela
Giglione
Diamond Proposal Number(s):
[12579]
Open Access
Abstract: The promising drug target N-myristoyltransferase (NMT) catalyses an essential protein modification thought to occur exclusively at N-terminal glycines (Gly). Here, we present high-resolution human NMT1 structures co-crystallised with reactive cognate lipid and peptide substrates, revealing high-resolution snapshots of the entire catalytic mechanism from the initial to final reaction states. Structural comparisons, together with biochemical analysis, provide unforeseen details about how NMT1 reaches a catalytically competent conformation in which the reactive groups are brought into close proximity to enable catalysis. We demonstrate that this mechanism further supports efficient and unprecedented myristoylation of an N-terminal lysine side chain, providing evidence that NMT acts both as N-terminal-lysine and glycine myristoyltransferase.
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Feb 2020
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9424]
Open Access
Abstract: Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27aMut, crystallized within 24 h and diffracted to 2.82 Å resolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by Phaser were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in Phaser. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in Phaser, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more suitable crystal forms.
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Mar 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Aurélie
Mousnier
,
Andrew S.
Bell
,
Dawid P.
Swieboda
,
Julia
Morales-Sanfrutos
,
Inmaculada
Perez-Dorado
,
James A.
Brannigan
,
Joseph
Newman
,
Markus
Ritzefeld
,
Jennie A.
Hutton
,
Anabel
Guedán
,
Amin S.
Asfor
,
Sean W.
Robinson
,
Iva
Hopkins-Navratilova
,
Anthony J.
Wilkinson
,
Sebastian L.
Johnston
,
Robin J.
Leatherbarrow
,
Tobias J.
Tuthill
,
Roberto
Solari
,
Edward W.
Tate
Diamond Proposal Number(s):
[12579, 7864, 9948]
Abstract: Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.
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May 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12579, 17221]
Open Access
Abstract: Cysteine-reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high-quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan-reactive compounds. Here we describe quantitative irreversible tethering (qIT), a general method for screening cysteine-reactive small molecules based upon the maximization of kinetic selectivity. We apply this method prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2-selective allosteric (type IV) kinase inhibitor whose novel mode-of-action could be exploited therapeutically.
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Feb 2018
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1221, 7864]
Open Access
Abstract: The parasite Plasmodium vivax is the most widely distributed cause of recurring malaria. N-Myristoyltransferase (NMT), an enzyme that catalyses the covalent attachment of myristate to the N-terminal glycine of substrate proteins, has been described as a potential target for the treatment of this disease. Herein, we report the synthesis and the structure-guided optimization of a series of quinolines with balanced activity against both Plasmodium vivax and Plasmodium falciparum N-myristoyltransferase (NMT).
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Nov 2016
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