I03-Macromolecular Crystallography
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Elizabeth R.
Allen
,
Stefanie A.
Krumm
,
Jayna
Raghwani
,
Steinar
Halldorsson
,
Angela
Elliott
,
Victoria A.
Graham
,
Elina
Koudriakova
,
Karl
Harlos
,
Daniel
Wright
,
George M.
Warimwe
,
Benjamin
Brennan
,
Juha T.
Huiskonen
,
Stuart D.
Dowall
,
Richard M.
Elliott
,
Oliver G.
Pybus
,
Dennis R.
Burton
,
Roger
Hewson
,
Katie J.
Doores
,
Thomas A.
Bowden
Diamond Proposal Number(s):
[14744]
Open Access
Abstract: The Gn subcomponent of the Gn-Gc assembly that envelopes the human and animal pathogen, Rift Valley fever virus (RVFV), is a primary target of the neutralizing antibody response. To better understand the molecular basis for immune recognition, we raised a class of neutralizing monoclonal antibodies (nAbs) against RVFV Gn, which exhibited protective efficacy in a mouse infection model. Structural characterization revealed that these nAbs were directed to the membrane-distal domain of RVFV Gn and likely prevented virus entry into a host cell by blocking fusogenic rearrangements of the Gn-Gc lattice. Genome sequence analysis confirmed that this region of the RVFV Gn-Gc assembly was under selective pressure and constituted a site of vulnerability on the virion surface. These data provide a blueprint for the rational design of immunotherapeutics and vaccines capable of preventing RVFV infection and a model for understanding Ab-mediated neutralization of bunyaviruses more generally.
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Dec 2018
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I03-Macromolecular Crystallography
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Abstract: Paired refinement is a procedure which analyses the impact of data from high
resolution on the quality of a refined structure model. Subsequent R-value analysis
leads to the determination of the optimal high resolution cutoff. In our particular
case of data from human receptor NKR-P1 extracellular domain, we could observe
differences in the paired refinement results in the case when the data were reintegrated
separately.
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Sep 2018
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Lucy C.
Walters
,
Karl
Harlos
,
Simon
Brackenridge
,
Daniel
Rozbesky
,
Jordan R.
Barrett
,
Vitul
Jain
,
Thomas S.
Walter
,
Chris A.
O’callaghan
,
Persephone
Borrow
,
Mireille
Toebes
,
Scott G.
Hansen
,
Jonah
Sacha
,
Shaheed
Abdulhaqq
,
Justin M.
Greene
,
Klaus
Früh
,
Emily
Marshall
,
Louis J.
Picker
,
E. Yvonne
Jones
,
Andrew J.
Mcmichael
,
Geraldine M.
Gillespie
Diamond Proposal Number(s):
[14744]
Open Access
Abstract: Through major histocompatibility complex class Ia leader sequence-derived (VL9) peptide binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccinations revealed contexts where HLA-E and the rhesus homologue, Mamu-E, presented diverse pathogen-derived peptides to CD8+ T cells, respectively. Here we present crystal structures of HLA-E in complex with HIV and Mtb-derived peptides. We show that despite the presence of preferred primary anchor residues, HLA-E-bound peptides can adopt alternative conformations within the peptide binding groove. Furthermore, combined structural and mutagenesis analyses illustrate a greater tolerance for hydrophobic and polar residues in the primary pockets than previously appreciated. Finally, biochemical studies reveal HLA-E peptide binding and exchange characteristics with potential relevance to its alternative antigen presenting function in vivo.
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Aug 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8423]
Abstract: LYPD6 is a Wnt signaling enhancer that promotes phosphorylation of the Wnt co‐receptor LRP6 (low‐density lipoprotein receptor‐related protein 6). It also binds the nicotinic acetylcholine receptor (nAChR). We report here the 1.25 Å resolution structure of the LYPD6 extracellular Ly6/uPAR (LU) domain and map its interaction with LRP6 by mutagenesis and surface plasmon resonance (SPR). The LYPD6LU structure reveals a “tri‐fingered protein domain” fold with the middle fingertip of the bearing a “NxI” motif, a tripeptide motif associated with LRP5/6 binding by Wnt inhibitors. Of the Ly6 protein family members, only LYPD6 has an NxI motif. Since mutations in the LYPD6 NxI motif abolish or severely reduce interaction with LRP6, our results indicate its key role in the interaction of LYPD6 with LRP6.
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Aug 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744]
Abstract: The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway. Whereas higher organisms derive folate from their diet and lack the enzymes for its synthesis, most eubacteria and a number of lower eukaryotes including malaria parasites synthesize tetrahydrofolate via DHPS. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by rise and spread of drug resistance mutations. Here, we present the crystal structure of PvHPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional PvHPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX’s effect on HPPK-DHPS is due to 4-amino benzoic acid (pABA) mimicry, and the PvHPPK-DHPS structure sheds light on the SDX-binding cavity as well as on mutations that effect SDX potency. We mapped five dominant drug resistance mutations in PvHPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the pABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/pABA/SDX interactions such that DHPS affinity for pABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the PvHPPK-DHPS structure rationalizes and unravels the structural bases for SDX resistance mutations and highlights architectural features in HPPK-DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites.
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Aug 2018
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I03-Macromolecular Crystallography
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Pronay
Das
,
Palak
Babbar
,
Nipun
Malhotra
,
Manmohan
Sharma
,
Gorakhnath R.
Jachak
,
Rajesh G.
Gonnade
,
Dhanasekaran
Shanmugam
,
Karl
Harlos
,
Manickam
Yogavel
,
Amit
Sharma
,
D. Srinivasa
Reddy
Diamond Proposal Number(s):
[14744]
Abstract: The dependence of drug potency on diastereomeric configurations is a key facet. Using a novel general divergent synthetic route for a three-chiral centre anti-malarial natural product cladosporin, we built its complete library of stereoisomers (cladologs) and assessed their inhibitory potential using parasite-, enzyme- and structure-based assays. We show that potency is manifest via tetrahyropyran ring conformations that are housed in the ribose binding pocket of parasite lysyl tRNA synthetase (KRS). Strikingly, drug potency between top and worst enantiomers varied 500-fold, and structures of KRS-cladolog complexes reveal that alterations at C3 and C10 are detrimental to drug potency where changes at C3 are sensed by rotameric flipping of Glutamate332. Given that scores of anti-malarial and anti-infective drugs contain chiral centers, this work provides a new foundation for focusing on inhibitor stereochemistry as a facet of anti-microbial drug development.
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May 2018
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Toxoplasma and Plasmodium are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The Toxoplasma gondii PLP1 (TgPLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of TgPLP1. The MACPF domain forms hexameric assemblies, with ring and helix geometries, and the APCβ domain has a novel β-prism fold joined to the MACPF domain by a short linker. Molecular dynamics simulations suggest that the helical MACPF oligomer preserves a biologically important interface, whereas the APCβ domain binds preferentially through a hydrophobic loop to membrane phosphatidylethanolamine, enhanced by the additional presence of inositol phosphate lipids. This mode of membrane binding is supported by site-directed mutagenesis data from a liposome-based assay. Together, these structural and biophysical findings provide insights into the molecular mechanism of membrane targeting by TgPLP1.
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Mar 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744]
Open Access
Abstract: Entry of enveloped viruses relies on insertion of hydrophobic residues of the viral fusion protein into the host cell membrane. However, the intermediate conformations during fusion remain unknown. Here, we address the fusion mechanism of Rift Valley fever virus. We determine the crystal structure of the Gn glycoprotein and fit it with the Gc fusion protein into cryo-electron microscopy reconstructions of the virion. Our analysis reveals how the Gn shields the hydrophobic fusion loops of the Gc, preventing premature fusion. Electron cryotomography of virions interacting with membranes under acidic conditions reveals how the fusogenic Gc is activated upon removal of the Gn shield. Repositioning of the Gn allows extension of Gc and insertion of fusion loops in the outer leaflet of the target membrane. These data show early structural transitions that enveloped viruses undergo during host cell entry and indicate that analogous shielding mechanisms are utilized across diverse virus families.
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Jan 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Hantaviruses are zoonotic pathogens with a near-global distribution that can cause severe hemorrhagic fever and pulmonary syndrome. The outer membrane of the hantavirus envelope displays a lattice of two glycoproteins, Gn and Gc, which orchestrate host cell recognition and entry. Here, we describe the crystal structure of the Gn glycoprotein ectodomain from the Asiatic Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Structural overlay analysis reveals that the HTNV Gn fold is highly similar to the Gn of Puumala virus (PUUV), a genetically and geographically distinct and less pathogenic hantavirus found predominantly in North-Eastern Europe, confirming that the hantaviral Gn fold is architecturally conserved across hantavirus clades. Interestingly, HTNV Gn crystallized at acidic pH, in a compact tetrameric configuration distinct from the organization at neutral pH. Analysis of the Gn, both in solution and in the context of the virion, confirms the pH-sensitive oligomeric nature of the glycoprotein, indicating that the hantaviral Gn undergoes structural transitions during host cell entry. These data allow us to present a structural model for how acidification during endocytic uptake of the virus triggers the dissociation of the metastable Gn-Gc lattice to enable insertion of the Gc-resident hydrophobic fusion loops into the host cell membrane. Together, these data reveal the dynamic plasticity of the structurally conserved hantaviral surface.
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Aug 2017
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I04-Macromolecular Crystallography
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Vitul
Jain
,
Manickam
Yogavel
,
Haruhisa
Kikuchi
,
Yoshiteru
Oshima
,
Norimitsu
Hariguchi
,
Makoto
Matsumoto
,
Preeti
Goel
,
Bastien
Touquet
,
Rajiv S.
Jumani
,
Fabienne
Tacchini-cottier
,
Karl
Harlos
,
Christopher D.
Huston
,
Mohamed-ali
Hakimi
,
Amit
Sharma
Diamond Proposal Number(s):
[14744]
Abstract: Developing anti-parasitic lead compounds that act on key vulnerabilities are necessary for new anti-infectives. Malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis and coccidiosis together kill >500,000 humans annually. Their causative parasites Plasmodium, Leishmania, Toxoplasma, Cryptosporidium and Eimeria display high conservation in many housekeeping genes, suggesting that these parasites can be attacked by targeting invariant essential proteins. Here, we describe selective and potent inhibition of prolyl-tRNA synthetases (PRSs) from the above parasites using a series of quinazolinone-scaffold compounds. Our PRS-drug co-crystal structures reveal remarkable active site plasticity that accommodates diversely substituted compounds, an enzymatic feature that can be leveraged for refining drug-like properties of quinazolinones on a per parasite basis. A compound we termed In-5 exhibited a unique double conformation, enhanced drug-like properties, and cleared malaria in mice. It thus represents a new lead for optimization. Collectively, our data offer insights into the structure-guided optimization of quinazolinone-based compounds for drug development against multiple human eukaryotic pathogens.
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Aug 2017
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