I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Nicole l.
Kallewaard
,
Davide
Corti
,
Patrick J.
Collins
,
Ursula
Neu
,
Josephine M.
Mcauliffe
,
Ebony
Benjamin
,
Leslie
Wachter-Rosati
,
Frances J.
Palmer-Hill
,
Andy Q.
Yuan
,
Philip A.
Walker
,
Matthias
Vorleander
,
Siro
Bianchi
,
Barbara
Guarino
,
Anna
De marco
,
Fabrizia
Vanzetta
,
Gloria
Agatic
,
Mathilde
Foglierini
,
Debora
Pinna
,
Blanca
Fernandez-Rodriguez
,
Alexander
Fruehwirth
,
Chiara
Silacci
,
Roksana W.
Ogrodowicz
,
Stephen R.
Martin
,
Federica
Sallusto
,
Joann A.
Suzich
,
Antonio
Lanzavecchia
,
Qing
Zhu
,
Steven J.
Gamblin
,
John J.
Skehel
Diamond Proposal Number(s):
[9826]
Open Access
Abstract: Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.
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Jul 2016
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I02-Macromolecular Crystallography
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Neil
Justin
,
Ying
Zhang
,
Cataldo
Tarricone
,
Stephen R.
Martin
,
Shuyang
Chen
,
Elizabeth
Underwood
,
Valeria
De Marco
,
Lesley F.
Haire
,
Philip A.
Walker
,
Danny
Reinberg
,
Jon R.
Wilson
,
Steven
Gamblin
Diamond Proposal Number(s):
[9826]
Open Access
Abstract: Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27me3) via its catalytic SET domain. A missense mutation in the substrate of PRC2, histone H3K27M, is associated with certain pediatric brain cancers and is linked to a global decrease of H3K27me3 in the affected cells thought to be mediated by inhibition of PRC2 activity. We present here the crystal structure of human PRC2 in complex with the inhibitory H3K27M peptide bound to the active site of the SET domain, with the methionine residue located in the pocket that normally accommodates the target lysine residue. The structure and binding studies suggest a mechanism for the oncogenic inhibition of H3K27M. The structure also reveals how binding of repressive marks, like H3K27me3, to the EED subunit of the complex leads to enhancement of the catalytic efficiency of the SET domain and thus the propagation of this repressive histone modification.
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Apr 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Xiaoli
Xiong
,
Davide
Corti
,
Junfeng
Liu
,
Debora
Pinna
,
Mathilde
Foglierini
,
Lesley J.
Calder
,
Stephen R.
Martin
,
Yi Pu
Lin
,
Phil
Walker
,
Patrick J.
Collins
,
Isabella
Monne
,
Amorsolo L.
Suguitan
,
Celia
Santos
,
Nigel J.
Temperton
,
Kanta
Subbarao
,
Antonio
Lanzavecchia
,
Steven
Gamblin
,
John J.
Skehel
Diamond Proposal Number(s):
[7707, 9826]
Open Access
Abstract: H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer’s formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.
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Jul 2015
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7707]
Open Access
Abstract: In 2004 an hemagglutinin 3 neuraminidase 8 (H3N8) equine influenza virus was transmitted from horses to dogs in Florida and subsequently spread throughout the United States and to Europe. To understand the molecular basis of changes in the antigenicity of H3 hemagglutinins (HAs) that have occurred during virus evolution in horses, and to investigate the role of HA in the equine to canine cross-species transfer, we used X-ray crystallography to determine the structures of the HAs from two antigenically distinct equine viruses and from a canine virus. Structurally all three are very similar with the majority of amino acid sequence differences between the two equine HAs located on the virus membrane-distal molecular surface. HAs of canine viruses are distinct in containing a Trp-222→Leu substitution in the receptor binding site that influences specificity for receptor analogs. In the fusion subdomain of canine and recent equine virus HAs a unique difference is observed by comparison with all other HAs examined to date. Analyses of site-specific mutant HAs indicate that a single amino acid substitution, Thr-30→Ser, influences interactions between N-terminal and C-terminal regions of the subdomain that are important in the structural changes required for membrane fusion activity. Both structural modifications may have facilitated the transmission of H3N8 influenza from horses to dogs.
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Jul 2014
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7707]
Abstract: H10N8 follows H7N9 and H5N1 as the latest in a line of avian influenza viruses that cause serious disease in humans and have become a threat to public health1. Since December 2013, three human cases of H10N8 infection have been reported, two of whom are known to have died. To gather evidence relating to the epidemic potential of H10 we have determined the structure of the haemagglutinin of a previously isolated avian H10 virus and we present here results relating especially to its receptor-binding properties, as these are likely to be major determinants of virus transmissibility. Our results show, first, that the H10 virus possesses high avidity for human receptors and second, from the crystal structure of the complex formed by avian H10 haemagglutinin with human receptor, it is clear that the conformation of the bound receptor has characteristics of both the 1918 H1N1 pandemic virus2 and the human H7 viruses isolated from patients in 2013 (ref. 3). We conclude that avian H10N8 virus has sufficient avidity for human receptors to account for its infection of humans but that its preference for avian receptors should make avian-receptor-rich human airway mucins4 an effective block to widespread infection. In terms of surveillance, particular attention will be paid to the detection of mutations in the receptor-binding site of the H10 haemagglutinin that decrease its avidity for avian receptor, and could enable it to be more readily transmitted between humans.
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Jul 2014
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Xiaoli
Xiong
,
Haixia
Xiao
,
Stephen R.
Martin
,
Peter J.
Coombs
,
Junfeng
Liu
,
Patrick
Collins
,
Sebastien
Vachieri
,
Phil
Walker
,
Yi Pu
Lin
,
John W.
Mccauley
,
Steven
Gamblin
,
John J.
Skehel
Diamond Proposal Number(s):
[7707]
Open Access
Abstract: Mutant H5N1 influenza viruses have been isolated from humans that have increased human receptor avidity. We have compared the receptor binding properties of these mutants with those of wild-type viruses, and determined the structures of their haemagglutinins in complex with receptor analogues. Mutants from Vietnam bind tighter to human receptor by acquiring basic residues near the receptor binding site. They bind more weakly to avian receptor because they lack specific interactions between Asn-186 and Gln-226. In contrast, a double mutant, Δ133/Ile155Thr, isolated in Egypt has greater avidity for human receptor while retaining wild-type avidity for avian receptor. Despite these increases in human receptor binding, none of the mutants prefers human receptor, unlike aerosol transmissible H5N1 viruses. Nevertheless, mutants with high avidity for both human and avian receptors may be intermediates in the evolution of H5N1 viruses that could infect both humans and poultry.
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May 2014
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Malaria is caused by a protozoan parasite that replicates within an intraerythrocytic parasitophorous vacuole. Release (egress) of malaria merozoites from the host erythrocyte is a highly regulated and calcium-dependent event that is critical for disease progression. Minutes before egress, an essential parasite serine protease called SUB1 is discharged into the parasitophorous vacuole, where it proteolytically processes a subset of parasite proteins that play indispensable roles in egress and invasion. Here we report the first crystallographic structure of Plasmodium falciparum SUB1 at 2.25 Å, in complex with its cognate prodomain. The structure highlights the basis of the calcium dependence of SUB1, as well as its unusual requirement for interactions with substrate residues on both prime and non-prime sides of the scissile bond. Importantly, the structure also reveals the presence of a solvent-exposed redox-sensitive disulphide bridge, unique among the subtilisin family, that likely acts as a regulator of protease activity in the parasite.
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May 2014
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7707]
Abstract: Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells1, 2 as well as resting CD4+ T cells3, 4 by reducing the cellular deoxynucleoside 5′-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function5, 6. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation7, 8. The molecular mechanism by which these viral proteins are able to usurp the host cell’s ubiquitination machinery to destroy the cell’s protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell’s normal protein degradation pathway to inactivate the cellular viral defence system.
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Dec 2013
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Bing
Xiao
,
Matthew J.
Sanders
,
David
Carmena
,
Nicola J.
Bright
,
Lesley F.
Haire
,
Elizabeth
Underwood
,
Bhakti R.
Patel
,
Richard B.
Heath
,
Phillip A.
Walker
,
Stefan
Hallen
,
Fabrizio
Giordanetto
,
Stephen R.
Martin
,
David
Carling
,
Steven
Gamblin
Diamond Proposal Number(s):
[7707]
Open Access
Abstract: AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.
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Dec 2013
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[7707]
Abstract: We report the structures of 4 sialoside receptor analogues complexed with H5 HA. The analogues included fucosylated and/or sulphated GlcNAc at position 3. Estimates of affinity of HA for the analogues were made by biolayer interferometry.
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Sep 2013
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