B21-High Throughput SAXS
B23-Circular Dichroism
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Philip
Bardelang
,
Ewan J.
Murray
,
Isobel
Blower
,
Sara
Zandomeneghi
,
Alice
Goode
,
Rohanah
Hussain
,
Divya
Kumari
,
Giuliano
Siligardi
,
Katsuaki
Inoue
,
Jeni
Luckett
,
James
Doutch
,
Jonas
Emsley
,
Weng C.
Chan
,
Philip
Hill
,
Paul
Williams
,
Boyan B.
Bonev
Diamond Proposal Number(s):
[5098, 12923, 13185, 13634, 15146]
Open Access
Abstract: Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.
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May 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26803]
Open Access
Abstract: Background: High molecular weight kininogen (HK) circulates in plasma as a complex with zymogen prekallikrein (PK). HK is both substrate and co-factor for activated plasma kallikrein (PKa) and the principal exosite interactions occur between PK N-terminal apple domains and the C-terminal D6 domain of HK. Objective: To determine the structure of the complex formed between PK apple domains and a HKD6 fragment and compare this to the FXI-HK complex. Methods: We produced recombinant FXI and PK heavy chains (HC) spanning all four apple domains. We co-crystallised PKHC (and subsequently FXIHC) with a 31 amino acid synthetic peptide spanning HK residues Ser565-Lys595 and determined the crystal structure. We also analysed the full length FXI-HK complex in solution using hydrogen deuterium exchange mass spectrometry (HDX-MS). Results and conclusions: The 2.3Å PKHC-HK peptide crystal structure revealed that the HKD6 sequence WIPDIQ (Trp569-Gln574) binds to the apple 1 domain and HK FNPISDFPDT (Phe582-Thr591) binds to the apple 2 domain with a flexible intervening sequence resulting in a bent double conformation. A second 3.2Å FXIHC-HK peptide crystal structure revealed a similar interaction with the apple 2 domain but an alternate, straightened conformation of the HK peptide where residues LSFN (Leu579-Asn583) interacts with a unique pocket formed between the apple 2 and 3 domains. HDX-MS of full length FXI-HK complex in solution confirmed interactions with both apple 2 and apple 3. The alternate conformations and exosite binding of the HKD6 peptide likely reflects the diverging relationship of HK to the functions of PK and FXI.
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Apr 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26803]
Abstract: Background: The platelet–signaling receptor glycoprotein VI (GPVI) is a promising antithrombotic target. We have previously raised a series of high-affinity nanobodies (Nbs) against GPVI and identified Nb2, Nb21, and Nb35 as potent GPVI inhibitors. The Nb2 binding site has been mapped to the D1 domain, which is directly adjacent to the CRP binding site. Ligand–binding complementary determining region 3 has only 15% conservation between all 3 Nbs. Objectives: To map the binding sites of Nb21 and Nb35 on GPVI. Methods: We determined the X-ray crystal structure of the D1 and D2 extracellular domains of the GPVI-Nb35 complex. We then looked at the effects of various GPVI mutations on the ability of Nbs to inhibit collagen binding and GPVI signaling using surface binding assays and transfected cell lines. Results: The crystal structure of GPVI bound to Nb35 was solved. GPVI was present as a monomer, and the D1+D2 conformation was comparable to that in the dimeric structure. Arg46, Tyr47, and Ala57 are common residues on GPVI targeted by both Nb2 and Nb35. Mutating Arg46 to an Ala abrogated the ability of Nb2, Nb21, and Nb35 to inhibit collagen–induced GPVI signaling and blocked the binding of all 3 Nbs. In addition, Arg60 was found to reduce Nb21 inhibition but not the inhibition Nb2 or Nb35. Conclusions: These findings reveal key residues involved in the high-affinity binding of GPVI inhibitors and negate the idea that GPVI dimerization induces a conformational change required for ligand binding.
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Dec 2022
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Von Willebrand factor (VWF) activates in response to shear flow to initiate hemostasis, while aberrant activation could lead to thrombosis. Above a critical shear force, the A1 domain of VWF becomes activated and captures platelets via the GPIb-IX complex. Here we show that the shear-responsive element controlling VWF activation resides in the discontinuous autoinhibitory module (AIM) flanking A1. Application of tensile force in a single-molecule setting induces cooperative unfolding of the AIM to expose A1. The AIM-unfolding force is lowered by truncating either N- or C-terminal AIM region, type 2B VWD mutations, or binding of a ristocetin-mimicking monoclonal antibody, all of which could activate A1. Furthermore, the AIM is mechanically stabilized by the nanobody that comprises caplacizumab, the only FDA-approved anti-thrombotic drug to-date that targets VWF. Thus, the AIM is a mechano-regulator of VWF activity. Its conformational dynamics may define the extent of VWF autoinhibition and subsequent activation under force.
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Apr 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: ADAMTS13 is a plasma metalloprotease that is essential for the regulation of von Willebrand factor (VWF) function, mediator of platelet recruitment to sites of blood vessel damage. ADAMTS13 function is dynamically regulated by structural changes induced by VWF binding that convert it from a latent to active conformation. ADAMTS13 global latency is manifest by the interaction of its C-terminal CUB1-2 domains with its central Spacer domain. We resolved the crystal structure of the ADAMTS13 CUB1-2 domains revealing a previously unreported configuration for the tandem CUB domains. Docking simulations between the CUB1-2 domains with the Spacer domain in combination with enzyme kinetic functional characterization of ADAMTS13 CUB domain mutants enabled the mapping of the CUB1-2 domain site that binds the Spacer domain. Together, these data reveal the molecular basis of the ADAMTS13 Spacer-CUB interaction and the control of ADAMTS13 global latency.
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Apr 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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I04-Macromolecular Crystallography
I23-Long wavelength MX
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Jean F. R.
Ribeiro
,
Lorenzo
Cianni
,
Chan
Li
,
Thomas G.
Warwick
,
Daniela
De Vita
,
Fabiana
Rosini
,
Fernanda
Dos Reis Rocho
,
Felipe G. P.
Martins
,
Peter W.
Kenny
,
Jeronimo
Lameira
,
Andrei
Leitão
,
Jonas
Emsley
,
Carlos A.
Montanari
Diamond Proposal Number(s):
[19880]
Abstract: Leishmania mexicana is an obligate intracellular protozoan parasite that causes the cutaneous form of leishmaniasis affecting South America and Mexico. The cysteine protease LmCPB is essential for the virulence of the parasite and therefore, it is an appealing target for antiparasitic therapy. A library of nitrile-based cysteine protease inhibitors was screened against LmCPB to develop a treatment of cutaneous leishmaniasis. Several compounds are sufficiently high-affinity LmCPB inhibitors to serve both as starting points for drug discovery projects and as probes for target validation. A 1.4 Å X-ray crystal structure, the first to be reported for LmCPB, was determined for the complex of this enzyme covalently bound to an azadipeptide nitrile ligand. Mapping the structure-activity relationships for LmCPB inhibition revealed superadditive effects for two pairs of structural transformations. Therefore, this work advances our understanding of azadipeptidyl and dipeptidyl nitrile structure-activity relationships for LmCPB structure-based inhibitor design. We also tested the same series of inhibitors on related cysteine proteases cathepsin L and Trypanosoma cruzi cruzain. The modulation of these mammalian and protozoan proteases represents a new framework for targeting papain-like cysteine proteases.
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Sep 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Rising numbers of cases of multidrug- and extensively drug-resistant Pseudomonas aeruginosa over recent years have created an urgent need for novel therapeutic approaches to cure potentially fatal infections. One such approach is virulence attenuation where anti-virulence compounds, designed to reduce pathogenicity without affording bactericidal effects, are employed to treat infections. P. aeruginosa uses the pqs quorum sensing (QS) system, to coordinate the expression of a large number of virulence determinants as well as bacterial-host interactions and hence represents an excellent anti-virulence target.
We report the synthesis and identification of a new series of thiazole-containing quinazolinones capable of inhibiting PqsR, the transcriptional regulator of the pqs QS system. The compounds demonstrated high potency (IC50 < 300 nM) in a whole-cell assay, using a mCTX::PpqsA-lux-based bioreporter for the P. aeruginosa PAO1-L and PA14 strains. Structural evaluation defined the binding modes of four analogues in the ligand-binding domain of PqsR through X-ray crystallography. Further work showed the ability of 6-chloro-3((2-pentylthiazol-4-yl)methyl)quinazolin-4(3H)-one (18) and 6-chloro-3((2-hexylthiazol-4-yl)methyl)quinazolin-4(3H)-one (19) to attenuate production of the PqsR-regulated virulence factor pyocyanin. Compounds 18 and 19 showed a low cytotoxic profile in the A549 human epithelial lung cell line making them suitable candidates for further pre-clinical evaluation.
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Aug 2020
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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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Fadi
Soukarieh
,
Ruiling
Liu
,
Manuel
Romero
,
Shaun N.
Roberston
,
William
Richardson
,
Simone
Lucanto
,
Eduard Vico
Oton
,
Naim Ruhul
Qudus
,
Alaa
Mashabi
,
Scott
Grossman
,
Sadiqur
Ali
,
Tomás
Sou
,
Irena
Kukavica-Ibrulj
,
Roger C.
Levesque
,
Christel A. S.
Bergström
,
Nigel
Halliday
,
Shailesh N.
Mistry
,
Jonas
Emsley
,
Stephan
Heeb
,
Paul
Williams
,
Miguel
Cámara
,
Michael J.
Stocks
Open Access
Abstract: Current treatments for Pseudomonas aeruginosa infections are becoming less effective because of the increasing rates of multi-antibiotic resistance. Pharmacological targeting of virulence through inhibition of quorum sensing (QS) dependent virulence gene regulation has considerable therapeutic potential. In P. aeruginosa, the pqs QS system regulates the production of multiple virulence factors as well as biofilm maturation and is a promising approach for developing antimicrobial adjuvants for combatting drug resistance. In this work, we report the hit optimisation for a series of potent novel inhibitors of PqsR, a key regulator of the pqs system, bearing a 2-((5-methyl-5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio) acetamide scaffold. The initial hit compound 7 (PAO1-L IC50 0.98 ± 0.02 μM, PA14 inactive at 10 μM) was obtained through a virtual screening campaign performed on the PqsR ligand binding domain using the University of Nottingham Managed Chemical Compound Collection. Hit optimisation gave compounds with enhanced potency against strains PAO1-L and PA14, evaluated using P. aeruginosa pqs-based QS bioreporter assays. Compound 40 (PAO1-L IC50 0.25 ± 0.12 μM, PA14 IC50 0.34 ± 0.03 μM) is one of the most potent PqsR antagonists reported showing significant inhibition of P. aeruginosa pyocyanin production and pqs system signaling in both planktonic cultures and biofilms. The co-crystal structure of 40 with the PqsR ligand binding domain revealed the specific binding interactions occurring between inhibitor and this key regulatory protein.
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May 2020
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