B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Lucy C.
Walters
,
Daniel
Rozbesky
,
Karl
Harlos
,
Max
Quastel
,
Hong
Sun
,
Sebastian
Springer
,
Robert P.
Rambo
,
Fiyaz
Mohammed
,
E. Yvonne
Jones
,
Andrew J.
Mcmichael
,
Geraldine M.
Gillespie
Diamond Proposal Number(s):
[19946]
Open Access
Abstract: MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.
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Jun 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yuguang
Zhao
,
William
Mahy
,
Nicky J.
Willis
,
Hannah L.
Woodward
,
David
Steadman
,
Elliott D.
Bayle
,
Benjamin N.
Atkinson
,
James
Sipthorp
,
Luca
Vecchia
,
Reinis R.
Ruza
,
Karl
Harlos
,
Fiona
Jeganathan
,
Stefan
Constantinou
,
Artur
Costa
,
Svend
Kjær
,
Magda
Bictash
,
Patricia C.
Salinas
,
Paul
Whiting
,
Jean-Paul
Vincent
,
Paul V.
Fish
,
E. Yvonne
Jones
Diamond Proposal Number(s):
[16814]
Open Access
Abstract: The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer’s disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 μM. Analysis of the fragments’ diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 μM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.
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Jun 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Abstract: Bicyclic azetidine compounds possess antimalarial activity via targeting of the cytoplasmic Plasmodium falciparum (Pf) protein translation enzyme phenylalanine-tRNA synthetase (cFRS). These drugs kill parasites both in vitro and in vivo, including the blood, liver, and transmission developmental stages. Here we present the co-crystal structure of PfcFRS with a potent inhibitor, the bicyclic azetidine BRD7929. Our studies reveal high-affinity binding of BRD7929 with PfcFRS along with exquisite specificity compared with the human enzyme, leading in turn to potent and selective inhibition of the parasite enzyme. Our co-crystal structure shows that BRD7929 binds in the active site in the α subunit of PfcFRS, where it occupies the amino acid site, an auxiliary site, and partially the ATP site. This structural snapshot of inhibitor-bound PfcFRS thus provides a platform for the structure-guided optimization of novel antimalarial compounds.
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Apr 2022
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I03-Macromolecular Crystallography
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Dapeng
Li
,
Simon
Brackenridge
,
Lucy C.
Walters
,
Olivia
Swanson
,
Karl
Harlos
,
Daniel
Rozbesky
,
Derek W.
Cain
,
Kevin
Wiehe
,
Richard M.
Scearce
,
Maggie
Barr
,
Zekun
Mu
,
Robert
Parks
,
Max
Quastel
,
Robert J.
Edwards
,
Yunfei
Wang
,
Wes
Rountree
,
Kevin O.
Saunders
,
Guido
Ferrari
,
Persephone
Borrow
,
E. Yvonne
Jones
,
S. Munir
Alam
,
Mihai L.
Azoitei
,
Geraldine M.
Gillespie
,
Andrew J.
Mcmichael
,
Barton F.
Haynes
Open Access
Abstract: The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.
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Mar 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744, 19946]
Open Access
Abstract: Transmission of the New World hemorrhagic fever arenaviruses Junín virus (JUNV) and Machupo virus (MACV) to humans is facilitated, in part, by the interaction between the arenavirus GP1 glycoprotein and the human transferrin receptor 1 (hTfR1). We utilize a mouse model of live-attenuated immunization with envelope exchange viruses to isolate neutralizing monoclonal antibodies (NAbs) specific to JUNV GP1 and MACV GP1. Structures of two NAbs, termed JUN1 and MAC1, demonstrate that they neutralize through disruption of hTfR1 recognition. JUN1 utilizes a binding mode common to all characterized infection- and vaccine-elicited JUNV-specific NAbs, which involves mimicking hTfR1 binding through the insertion of a tyrosine into the receptor-binding site. In contrast, MAC1 undergoes a tyrosine-mediated mode of antigen recognition distinct from that used by the reported anti-JUNV NAbs and the only other characterized anti-MACV NAb. These data reveal the varied modes of GP1-specific recognition among New World arenaviruses by the antibody-mediated immune response.
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Mar 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Abstract: Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS) represent a promising therapeutic anti‐malarial target. Cladosporin, a tool compound was identified as a selective and potent PfKRS inhibitor but lacks metabolic stability. Here, we report chemical synthesis, biological evaluation and structural characterization of analogues where the tetrahydropyran (THP) frame of cladosporin is replaced with the piperidine ring bearing functional group variations. Thermal binding, enzymatic, kinetic and parasitic assays complemented with x‐ray crystallography reveals compounds to be of moderate potency. Co‐crystals of Cla‐B and Cla‐C with PfKRS reveals key atomic configurations that allow drug binding to, and inhibition of the enzyme. Collectively, these piperidine ring scaffold inhibitors lays a framework for further structural editing and functional modifications of cladosporin scaffold to obtain a potent lead.
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May 2021
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I24-Microfocus Macromolecular Crystallography
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Manmohan
Sharma
,
Nipun
Malhotra
,
Manickam
Yogavel
,
Karl
Harlos
,
Bruno
Melillo
,
Eamon
Comer
,
Arthur
Gonse
,
Suhel
Parvez
,
Branko
Mitasev
,
Francis G.
Fang
,
Stuart L.
Schreiber
,
Amit
Sharma
Diamond Proposal Number(s):
[19946]
Open Access
Abstract: The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.
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Jan 2021
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Alice J.
Bochel
,
Christopher
Williams
,
Airlie J.
Mccoy
,
Hans-Jürgen
Hoppe
,
Ashley J.
Winter
,
Ryan D.
Nicholls
,
Karl
Harlos
,
E. Yvonne
Jones
,
Imre
Berger
,
A. Bassim
Hassan
,
Matthew P.
Crump
Diamond Proposal Number(s):
[8423]
Abstract: The cation-independent mannose 6-phosphate (M6P)/Insulin-like growth factor-2 receptor (CI-MPR/IGF2R) is an ∼300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and internalizing IGF2. The extracellular region of the CI-MPR has 15 homologous domains, including M6P-binding domains (D) 3, 5, 9, and 15 and IGF2-binding domain 11. We have focused on solving the first structures of human D7–10 within two multi-domain constructs, D9–10 and D7–11, and provide the first high-resolution description of the high-affinity M6P-binding D9. Moreover, D9 stabilizes a well-defined hub formed by D7–11 whereby two penta-domains intertwine to form a dimeric helical-type coil via an N-glycan bridge on D9. Remarkably the D7–11 structure matches an IGF2-bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues may impart receptor rigidity and play a role in structural transitions at low pH.
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Sep 2020
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
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Ondřej
Skořepa
,
Samuel
Pazicky
,
Barbora
Kalousková
,
Jan
Bláha
,
Celeste
Abreu
,
Tomáš
Ječmen
,
Michal
Rosůlek
,
Alexander
Fish
,
Arthur
Sedivy
,
Karl
Harlos
,
Jan
Dohnalek
,
Tereza
Skálová
,
Ondřej
Vaněk
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI− cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.
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Jul 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell‐attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. We have discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. Our high‐resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities.
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Jun 2020
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