I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[14891]
Abstract: Immunomodulatory drug (IMiD) resistance is a key clinical challenge in myeloma treatment. Previous data suggests almost one third of myeloma patients acquire mutations in the key IMiD effector cereblon by the time they are pomalidomide refractory. Some events, including stop codons/frameshift mutations and copy loss, having clearly explicable effects on cereblon function. Missense mutations have also been reported throughout the length of cereblon but their functional impact has not been systematically studied. This study modelled selected missense mutations and examined their effect on cereblon function also analysing whether any mutations deleterious to IMiD action could be overcome using the novel cereblon binding agents (CELMoDs). Three patterns of response to missense mutations were apparent, mutations that led to complete loss of CRBN function for all agents, those that had no effect on CRBN function and those with agent-dependent effect on CRBN function. The latter group of 4 mutations were profiled in more detail with confirmatory experiments demonstrating an ability of the more potent CELMoDs to lead to neosubstrate degradation and cell death even though IMiDs were not active. Dynamic modelling based on a newly generated crystal structure of the DDB1/CRBN/lenalidomide complex, with greater resolution than those published to date, helped to understand the impact of these mutations. These results have important implications for the interpretation of CRBN sequencing results from patients for future therapy decisions, particularly differentiating those who may, despite relapsing on IMiDs with CRBN mutations, have the potential to still benefit from the use of CELMoD agents.
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Jan 2025
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I24-Microfocus Macromolecular Crystallography
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Nicholas A.
Arce
,
Zoe
Markham-Lee
,
Qian
Liang
,
Shabir
Najmudin
,
Emily R.
Legan
,
Gabrielle
Dean
,
Ally J.
Su
,
Moriah S.
Wilson
,
Robert F.
Sidonio
,
Pete
Lollar
,
Jonas
Emsley
,
Renhao
Li
Diamond Proposal Number(s):
[19880]
Abstract: Activation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify two groups of nanobodies. One group, represented by clone 6D12, is conformation-insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1. 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation-sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and that binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.
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Jan 2024
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Sara
Canovas Nunes
,
Serena
De Vita
,
Andrew
Anighoro
,
François
Autelitano
,
Edward
Beaumont
,
Pamela
Klingbeil
,
Meaghan
Mcguinness
,
Beatrice
Duvert
,
Chad
Harris
,
Lu
Yang
,
Sheela Pangeni
Pokharel
,
Chun-Wei
Chen
,
Monika
Ermann
,
David A.
Williams
,
Haiming
Xu
Open Access
Abstract: RAS mutations prevalent in high-risk leukemia have been linked to relapse and chemotherapy resistance. Efforts to directly target RAS proteins have been largely unsuccessful. However, since RAS-mediated transformation is dependent on signaling through the RAS-related C3 botulinum toxin substrate (RAC) small GTPase, we hypothesized that targeting RAC may be an effective therapeutic approach in RAS mutated tumors. Here we describe multiple small molecules capable of inhibiting RAC activation in acute lymphoblastic leukemia cell lines. One of these, DW0254, also demonstrates promising anti-leukemic activity in RAS-mutated cells. Using chemical proteomics and biophysical methods, we identified the hydrophobic pocket of phosphodiester 6 subunit delta (PDE6D), a known RAS chaperone, as a target for this compound. Inhibition of RAS localization to the plasma membrane upon DW0254 treatment is associated with RAC inhibition through a phosphatidylinositol-3-kinase/AKT-dependent mechanism. Our findings provide new insights into the importance of PDE6D-mediated transport for RAS-dependent RAC activation and leukemic cell survival.
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Apr 2022
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Cavan
Bennett
,
Moyra
Lawrence
,
Jose A.
Guerrero
,
Simon
Stritt
,
Amie K.
Waller
,
Yahui
Yan
,
Richard W.
Mifsud
,
Jose
Ballester-Beltran
,
Ayesha A.
Baig
,
Annett
Mueller
,
Louisa
Mayer
,
James
Warland
,
Christopher J.
Penkett
,
Parsa
Akbari
,
Thomas
Moreau
,
Amanda L
Evans
,
Souradip
Mookerjee
,
Gary J.
Hoffman
,
Kourosh
Saeb-Parsy
,
David
Adams
,
Amber L
Couzens
,
Markus
Bender
,
Wendy N.
Erber
,
Bernhard
Nieswandt
,
Randy J.
Read
,
Cedric
Ghevaert
Open Access
Abstract: The process of platelet production has so far been understood to be a two-stage process: megakaryocyte (MK) maturation from haematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases “beads-on-a-string” preplatelets into the blood stream that undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25-48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly due to increased microtubule glutamylation via CRLF3’s interaction with key members of the Hippo pathway. Using a mouse model of JAK2V617F Essential Thrombocythaemia (ET), we show that a lack of CRLF3 leads to a long-term lineage-specific normalisation of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythaemia.
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Jan 2022
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I04-Macromolecular Crystallography
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Giorgia
Chiodin
,
Joel D.
Allen
,
Dean J.
Bryant
,
Philip
Rock
,
Enrica A.
Martino
,
Beatriz
Valle-Argos
,
Patrick J.
Duriez
,
Yasunori
Watanabe
,
Isla
Henderson
,
James S.
Blachly
,
Katy J.
Mccann
,
Jonathan C.
Strefford
,
Graham
Packham
,
Teunis B. H.
Geijtenbeek
,
Carl G.
Figdor
,
George W.
Wright
,
Louis M.
Staudt
,
Richard
Burack
,
Thomas A.
Bowden
,
Max
Crispin
,
Freda K.
Stevenson
,
Francesco
Forconi
Diamond Proposal Number(s):
[14744]
Abstract: Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma–associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell–like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell–like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell–specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN–expressing M2-polarized macrophages.
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Oct 2021
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I04-Macromolecular Crystallography
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Alexandre
Slater
,
Ying
Di
,
Joanne C
Clark
,
Natalie Jasmin
Jooss
,
Eleyna M
Martin
,
Fawaz Obaidullah
Alenazy
,
Mark R.
Thomas
,
Robert A. S.
Ariëns
,
Andrew B.
Herr
,
Natalie S.
Poulter
,
Jonas
Emsley
,
Stephen P.
Watson
Diamond Proposal Number(s):
[19880]
Abstract: GPVI is the major signalling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 (CDR3) loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterised their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nanobodies from six different binding classes showed the strongest binding to recombinant GPVI-Fc suggesting that there was not a single dominant class. The most potent three, Nb2, 21 and 35, inhibited collagen-induced platelet aggregation with nanomolar IC50 values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the CRP binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signalling was abolished in a cell-based NFAT-reporter assay. This demonstrates that the C-C' loop region is important for GPVI signalling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.
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Jan 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Abstract: The contact system is composed of Factor XII (FXII), prekallikrein (PK) and co-factor kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+ dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion with residues Arg36 and Arg65 forming contacts with two distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII binding site and a comparison with the ligand free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+ binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with SPR demonstrate the gC1qR Zn2+ site contributes to FXII binding and plasma based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only one high affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher order 500kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors which may be a prelude for initiating the cascades which drive bradykinin generation and the intrinsic pathway of coagulation.
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Jun 2020
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I24-Microfocus Macromolecular Crystallography
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Juliet
Morgan
,
Muhammad
Saleem
,
Ruiqi
Ng
,
Caroline
Armstrong
,
Szu S.
Wong
,
Simon G.
Caulton
,
Alice
Fickling
,
Huw E. L.
Williams
,
Adam D.
Munday
,
José A.
López
,
Mark S.
Searle
,
Jonas
Emsley
Abstract: Cell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, αMβ2, CD11b/CD18) and platelet glycoprotein Ibα (GPIbα) are critical to vascular inflammation. To define the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIbα N-terminal domain (GPIbαN) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIbαN and Mac-1 I-domain to 2 Å and 2.5 Å resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the α7-helix with a glutamate side chain completing the octahedral coordination sphere of the MIDAS Mg2+ ion. The amino acid sequence of the α7-helix and disposition of the glutamic acid matches the C-terminal capping region α-helix of GPIbα effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIbα leucine-rich repeat (LRR) capping α-helix coordinates directly to the Mac-1 MIDAS Mg2+ ion. The Mac-1:GPIbαN complex involves additional interactions consolidated by an elongated pocket flanking the GPIbαN LRR capping α-helix. The GPIbαN α-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIbαN. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, confirmed the importance of GPIbαN residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex.
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May 2019
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.
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Mar 2017
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692, 10369]
Abstract: Factor XI (FXI) is the zymogen of factor XIa (FXIa) which cleaves factor IX in the intrinsic pathway of coagulation. FXI is known to exist as a dimer and form interactions with multiple proteins via its four apple domains in the "saucer section" of the enzyme however to date, no complex crystal structure has been described. To investigate protein interactions of FXI a large random peptide library consisting of 106-107 peptides was screened for FXI binding and this identified a series of FXI binding motifs containing the signature Asp-Phe-Pro (DFP) tripeptide. Motifs containing this core tripeptide were found in diverse proteins including the known ligand high molecular weight kininogen (HK) as well as extracellular matrix proteins laminin and collagen V. To define the binding site on FXI we determined the crystal structure of FXI in complex with the HK derived peptide NPISDFPDT. This revealed the location of the DFP peptide bound to the FXI apple 2 domain and central to the interaction the DFP phenylalanine side chain inserts into a major hydrophobic pocket in the apple 2 domain and the isoleucine occupies a flanking minor pocket. Two further structures of FXI in complex with the laminin derived peptide EFPDFP and a DFP peptide from the random screen demonstrated binding in the same pocket although in a slightly different conformation, thus revealing some flexibility in the molecular interactions of the FXI apple 2 domain.
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Mar 2016
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