I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17201]
Open Access
Abstract: N-utilization substance A (NusA) is a regulatory factor with pleiotropic functions in gene expression in bacteria. Archaea encode two conserved small proteins, NusA1 and NusA2, with domains orthologous to the two RNA binding K Homology (KH) domains of NusA. Here, we report the crystal structures of NusA2 from Sulfolobus acidocaldarius and Saccharolobus solfataricus obtained at 3.1 Å and 1.68 Å, respectively. NusA2 comprises an N-terminal zinc finger followed by two KH-like domains lacking the GXXG signature. Despite the loss of the GXXG motif, NusA2 binds single-stranded RNA. Mutations in the zinc finger domain compromise the structural integrity of NusA2 at high temperatures and molecular dynamics simulations indicate that zinc binding provides an energy barrier preventing the domain from reaching unfolded states. A structure-guided phylogenetic analysis of the KH-like domains supports the notion that the NusA2 clade is ancestral to the ribosomal protein eS7 in eukaryotes, implying a potential role of NusA2 in translation.
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Nov 2024
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I23-Long wavelength MX
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Diamond Proposal Number(s):
[30393, 2322]
Open Access
Abstract: The identification of multiple simultaneous orientations of small molecule inhibitors binding to a protein target is a common challenge. It has recently been reported that the conformational heterogeneity of ligands is widely underreported in the Protein Data Bank, which is likely to impede optimal exploitation to improve affinity of these ligands. Significantly less is even known about multiple binding orientations for fragments (<300 Da), although this information would be essential for subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here, we use recently reported fragment hits for the SARS-CoV-2 non-structural protein 1 (nsp1) N-terminal domain to propose a general procedure for unambiguously identifying binding orientations of 2-dimensional fragments containing either sulphur or chloro substituents within the wavelength range of most tunable beamlines. By measuring datasets at two energies, using a tunable beamline operating in vacuum and optimised for data collection at very low X-ray energies, we show that the anomalous signal can be used to identify multiple orientations in small fragments containing sulphur and/or chloro substituents or to verify recently reported conformations. Although in this specific case we identified the positions of sulphur and chlorine in fragments bound to their protein target, we are confident that this work can be further expanded to additional atoms or ions which often occur in fragments. Finally, our improvements in the understanding of binding orientations will also serve to improve the rational optimisation of SARS-CoV-2 nsp1 fragment hits.
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Jul 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Grace Q.
Gong
,
Benoit
Bilanges
,
Ben
Allsop
,
Glenn
Masson
,
Victoria
Roberton
,
Trevor
Askwith
,
Sally
Oxenford
,
Ralitsa R.
Madsen
,
Sarah E.
Conduit
,
Dom
Bellini
,
Martina
Fitzek
,
Matt
Collier
,
Osman
Najam
,
Zhenhe
He
,
Ben
Wahab
,
Stephen H.
Mclaughlin
,
A. W. Edith
Chan
,
Isabella
Feierberg
,
Andrew
Madin
,
Daniele
Morelli
,
Amandeep
Bhamra
,
Vanesa
Vinciauskaite
,
Karen E.
Anderson
,
Silvia
Surinova
,
Nikos
Pinotsis
,
Elena
Lopez-Guadamillas
,
Matthew
Wilcox
,
Alice
Hooper
,
Chandni
Patel
,
Maria A.
Whitehead
,
Tom D.
Bunney
,
Len R.
Stephens
,
Phillip T.
Hawkins
,
Matilda
Katan
,
Derek M.
Yellon
,
Sean M.
Davidson
,
David M.
Smith
,
James B.
Phillips
,
Richard
Angell
,
Roger L.
Williams
,
Bart
Vanhaesebroeck
Diamond Proposal Number(s):
[28677]
Abstract: Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia–reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
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May 2023
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Lorena
Zuliani-Alvarez
,
Morten L.
Govasli
,
Jane
Rasaiyaah
,
Chris
Monit
,
Stephen O.
Perry
,
Rebecca P.
Sumner
,
Simon
Mcalpine-Scott
,
Claire
Dickson
,
K. M.
Rifat Faysal
,
Laura
Hilditch
,
Richard J.
Miles
,
Frederic
Bibollet-Ruche
,
Beatrice H.
Hahn
,
Till
Boecking
,
Nikos
Pinotsis
,
Leo C.
James
,
David A.
Jacques
,
Greg J.
Towers
Diamond Proposal Number(s):
[8547, 11235]
Open Access
Abstract: Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.
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Nov 2022
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23853, 30393]
Open Access
Abstract: The regular reappearance of coronavirus (CoV) outbreaks over the past 20 years has caused significant health consequences and financial burdens worldwide. The most recent and still ongoing novel CoV pandemic, caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has brought a range of devastating consequences. Due to the exceptionally fast development of vaccines, the mortality rate of the virus has been curbed to a significant extent. However, the limitations of vaccination efficiency and applicability, coupled with the still high infection rate, emphasise the urgent need for discovering safe and effective antivirals against SARS-CoV-2 by suppressing its replication or attenuating its virulence. Non-structural protein 1 (nsp1), a unique viral and conserved leader protein, is a crucial virulence factor for causing host mRNA degradation, suppressing interferon (IFN) expression and host antiviral signalling pathways. In view of the essential role of nsp1 in the CoV life cycle, it is regarded as an exploitable target for antiviral drug discovery. Here, we report a variety of fragment hits against the N-terminal domain of SARS-CoV-2 nsp1 identified by fragment-based screening via X-ray crystallography. We also determined the structure of nsp1 at atomic resolution (0.99 Å). Binding affinities of hits against nsp1 and potential stabilisation were determined by orthogonal biophysical assays such as microscale thermophoresis and thermal shift assays. We identified two ligand-binding sites on nsp1, one deep and one shallow pocket, which are not conserved between the three medically relevant SARS, SARS-CoV-2 and MERS coronaviruses. Our study provides an excellent starting point for the development of more potent nsp1-targeting inhibitors and functional studies on SARS-CoV-2 nsp1.
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Oct 2022
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I03-Macromolecular Crystallography
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Nikos
Pinotsis
,
Karolina
Zielinska
,
Mrigya
Babuta
,
Joan L.
Arolas
,
Julius
Kostan
,
Muhammad Bashir
Khan
,
Claudia
Schreiner
,
Anita
Salmazo
,
Luciano
Ciccarelli
,
Martin
Puchinger
,
Eirini A.
Gkougkoulia
,
Euripedes De Almeida
Ribeiro
,
Thomas C.
Marlovits
,
Alok
Bhattacharya
,
Kristina
Djinovic-Carugo
Abstract: The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin–binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca2+ in nonmuscle cells. Here we report the mechanism of Ca2+-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca2+-free and Ca2+-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca2+, binding of which can only be regulated in the presence of physiological concentrations of Mg2+. Ca2+ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin–binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery.
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Sep 2020
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[14704]
Open Access
Abstract: The organization of the mitochondrial electron transport chain proteins into supercomplexes (SCs) is now undisputed; however, their assembly process, or the role of differential expression isoforms, remain to be determined. In Saccharomyces cerevisiae, cytochrome c oxidase (CIV) forms SCs of varying stoichiometry with cytochrome bc1 (CIII). Recent studies have revealed, in normoxic growth conditions, an interface made exclusively by Cox5A, the only yeast respiratory protein that exists as one of two isoforms depending on oxygen levels. Here we present the cryo-EM structures of the III2-IV1 and III2-IV2 SCs containing the hypoxic isoform Cox5B solved at 3.4 and 2.8 Å, respectively. We show that the change of isoform does not affect SC formation or activity, and that SC stoichiometry is dictated by the level of CIII/CIV biosynthesis. Comparison of the CIV5B- and CIV5A-containing SC structures highlighted few differences, found mainly in the region of Cox5. Additional density was revealed in all SCs, independent of the CIV isoform, in a pocket formed by Cox1, Cox3, Cox12, and Cox13, away from the CIII–CIV interface. In the CIV5B-containing hypoxic SCs, this could be confidently assigned to the hypoxia-induced gene 1 (Hig1) type 2 protein Rcf2. With conserved residues in mammalian Hig1 proteins and Cox3/Cox12/Cox13 orthologs, we propose that Hig1 type 2 proteins are stoichiometric subunits of CIV, at least when within a III-IV SC.
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Apr 2020
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[14704]
Abstract: Cytochrome c oxidase (complex IV, CIV) is known in mammals to exist independently or in association with other respiratory proteins to form supercomplexes (SCs). In Saccharomyces cerevisiae, CIV is found solely in an SC with cytochrome bc1 (complex III, CIII). Here, we present the cryogenic electron microscopy (cryo-EM) structure of S. cerevisiae CIV in a III2IV2 SC at 3.3 Å resolution. While overall similarity to mammalian homologs is high, we found notable differences in the supernumerary subunits Cox26 and Cox13; the latter exhibits a unique arrangement that precludes CIV dimerization as seen in bovine. A conformational shift in the matrix domain of Cox5A—involved in allosteric inhibition by ATP—may arise from its association with CIII. The CIII–CIV arrangement highlights a conserved interaction interface of CIII, albeit one occupied by complex I in mammalian respirasomes. We discuss our findings in the context of the potential impact of SC formation on CIV regulation.
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Dec 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9204]
Open Access
Abstract: Legionnaires disease is a severe form of pneumonia caused by the bacterium Legionella pneumophila. L. pneumophila pathogenicity relies on secretion of more than 300 effector proteins by a type IVb secretion system. Among these Legionella effectors, WipA has been primarily studied because of its dependence on a chaperone complex, IcmSW, for translocation through the secretion system, but its role in pathogenicity has remained unknown. In this study, we present the crystal structure of a large fragment of WipA, WipA435. Surprisingly, this structure revealed a serine/threonine phosphatase fold that unexpectedly targets tyrosine-phosphorylated peptides. The structure also revealed a sequence insertion that folds into an α-helical hairpin, the tip of which adopts a canonical coiled-coil structure. The purified protein was a dimer, whose dimer interface involves interactions between the coiled-coil of one WipA molecule and the phosphatase domain of another. Given the ubiquity of protein-protein interaction mediated by interactions between coiled-coils, we hypothesize that WipA can thereby transition from a homo-dimeric state to a hetero-dimeric state in which the coiled-coil region of WipA is engaged in a protein-protein interaction with a tyrosine-phosphorylated host target. In conclusion, these findings help advance our understanding of the molecular mechanisms of an effector involved in Legionella virulence and may inform approaches to elucidate the function of other effectors
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Apr 2017
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