I03-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Diamond Proposal Number(s):
[29990, 36446]
Open Access
Abstract: The red fluorescent protein mCherry is one of the most widely used fluorescent proteins in biology. Here, we have changed the chromophore chemistry by converting the thioether group of M66 to a thiol group through mutation to cysteine. The new variant, termed mCoral (due to its orange fluorescence hue), has similar brightness to mCherry but improved resistance to hydrogen peroxide. The variant is also responsive to pH with low and high pKa forms that have distinct spectral properties, which DFT analysis suggests is due to protonation state changes in the newly introduced thiol group, as well as the phenol group. The structure of mCoral reveals that the M66C mutation creates a space within the β-barrel structure that is filled by a water molecule, which makes new polar interactions, including the backbone carbonyl group of F65. Molecular dynamics simulations suggest that this additional water molecule, together with local solvation around the chromophore, could play a role in promoting planarity of the full conjugated system comprising the chromophore. The mCoral chromophore makes slightly more H-bonds with water than mCherry. The main water exit point for mCherry is also narrower in mCoral, providing a potential explanation for increased resistance to hydrogen peroxide. Overall, a small structural change to mCherry has resulted in a new fluorescent protein with potentially useful characteristics and an insight into the role of dynamics and water in defining the structure–function relationship in red fluorescent proteins.
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Dec 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18812]
Open Access
Abstract: Here, we have linked one of the most common protein-protein interaction events, homodimerisation, to an essential trace metal, copper, through engineering green fluorescent protein. Mutation of H148 to cysteine promotes the neutral phenolic chromophore in the monomer that excites predominantly at ~400 nm. Homodimerisation via a copper-dependent disulphide bridge, switches the chromophore to the charged phenolate that excites at ~490 nm. The result is ~30 fold increase in the fluorescence emission ratio. Homo-dimerisation kinetics are further improved by optimising the sfGFP homodimer interface, generating the variant termed GFP-diS2. Structures of the monomeric and dimeric GFP-diS2 suggests charge switching is through peptide bond flipping and the formation of a buried organised water network around the chromophore that span the interface region. Fusion to a leucine zipper protein dimerisation element greatly increased GFP-diS2 association rate making it a more effective copper sensor in vitro and in vivo with Cu(I) instigating the signal change quicker and at lower ion concentrations than Cu(II). Thus, GFP-diS2 provides the framework for generating a sensitive genetically encoded copper sensor and will eventually be adapted to monitor one of the most important protein-protein interactions in biology, homo-oligomerisation.
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Oct 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Garry
Dolton
,
Anna
Bulek
,
Aaron
Wall
,
Hannah
Thomas
,
Jade R.
Hopkins
,
Cristina
Rius
,
Sarah A. E.
Galloway
,
Thomas
Whalley
,
Li Rong
Tan
,
Théo
Morin
,
Nader
Omidvar
,
Anna
Fuller
,
Katie
Topley
,
Md Samiul
Hasan
,
Shikha
Jain
,
Nirupa
D’souza
,
Thomas
Hodges-Hoyland
,
Owen B.
Spiller
,
Deborah
Kronenberg-Versteeg
,
Barbara
Szomolay
,
Hugo A.
Van Den Berg
,
Lucy C.
Jones
,
Mark
Peakman
,
David K.
Cole
,
Pierre J.
Rizkallah
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[10462, 18812]
Open Access
Abstract: CD8+ T cells destroy insulin-producing pancreatic β cells in type 1 diabetes through HLA class I–restricted presentation of self-antigens. Combinatorial peptide library screening was used to produce a preferred peptide recognition landscape for a patient-derived T cell receptor (TCR) that recognized the preproinsulin-derived (PPI-derived) peptide sequence LWMRLLPLL in the context of disease risk allele HLA A*24:02. Data were used to generate a strong superagonist peptide, enabling production of an autoimmune HLA A*24:02–peptide–TCR structure by crystal seeding. TCR binding to the PPI epitope was strongly focused on peptide residues Arg4 and Leu5, with more flexibility at other positions, allowing the TCR to strongly engage many peptides derived from pathogenic bacteria. We confirmed an epitope from Klebsiella that was recognized by PPI-reactive T cells from 3 of 3 HLA A*24:02+ patients. Remarkably, the same epitope selected T cells from 7 of 8 HLA A*24+ healthy donors that cross-reacted with PPI, leading to recognition and killing of HLA A*24:02+ cells expressing PPI. These data provide a mechanism by which molecular mimicry between pathogen and self-antigens could have resulted in the breaking of self-tolerance to initiate disease.
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Sep 2024
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I03-Macromolecular Crystallography
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Sarah
Hulin-Curtis
,
James K.
Geary
,
Bruce J.
Maclachlan
,
Danny M.
Altmann
,
Laury
Baillon
,
David K.
Cole
,
Alexander
Greenshields-Watson
,
Sophie J.
Hesketh
,
Ian R.
Humphreys
,
Ian M.
Jones
,
Sarah N.
Lauder
,
Georgina H.
Mason
,
Kathryn
Smart
,
D. Oliver
Scourfield
,
Jake
Scott
,
Ksenia
Sukhova
,
Richard J.
Stanton
,
Aaron
Wall
,
Pierre J.
Rizkallah
,
Wendy S.
Barclay
,
Awen
Gallimore
,
Andrew
Godkin
Diamond Proposal Number(s):
[10462]
Open Access
Abstract: CD4+ T cells are central to adaptive immunity. Their role in cross-protection in viral infections such as influenza and severe acute respiratory syndrome (SARS) is well documented; however, molecular rules governing T cell receptor (TCR) engagement of peptide-human leukocyte antigen (pHLA) class II are less understood. Here, we exploit an aspect of HLA class II presentation, the peptide-flanking residues (PFRs), to “tune” CD4+ T cell responses within an in vivo model system of influenza. Using a recombinant virus containing targeted substitutions at immunodominant HLA-DR1 epitopes, we demonstrate limited weight loss and improved clinical scores after heterosubtypic re-challenge. We observe enhanced protection linked to lung-derived influenza-specific CD4+ and CD8+ T cells prior to re-infection. Structural analysis of the ternary TCR:pHLA complex identifies that flanking amino acids influence side chains in the core 9-mer peptide, increasing TCR affinity. Augmentation of CD4+ T cell immunity is achievable with a single mutation, representing a strategy to enhance adaptive immunity that is decoupled from vaccine modality.
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Jun 2024
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18812, 20147, 29990]
Open Access
Abstract: Human adenoviruses (HAdV) are widespread pathogens causing usually mild infections. The Species D (HAdV-D) cause gastrointestinal tract infections and epidemic keratoconjunctivitis (EKC). Despite being significant pathogens, knowledge around HAdV-D mechanism of cell infection is lacking. Sialic acid (SA) usage has been proposed as a cell infection mechanism for EKC causing HAdV-D. Here we highlight an important role for SA engagement by many HAdV-D. We provide apo state crystal structures of 7 previously undetermined HAdV-D fiber-knob proteins, and structures of HAdV-D25, D29, D30 and D53 fiber-knob proteins in complex with SA. Biologically, we demonstrate that removal of cell surface SA reduced infectivity of HAdV-C5 vectors pseudotyped with HAdV-D fiber-knob proteins, whilst engagement of the classical HAdV receptor CAR was variable. Our data indicates variable usage of SA and CAR across HAdV-D. Better defining these interactions will enable improved development of antivirals and engineering of the viruses into refined therapeutic vectors.
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Sep 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yuan
Chen
,
Georgina H.
Mason
,
D. Oliver
Scourfield
,
Alexander
Greenshields-Watson
,
Tracey A.
Haigh
,
Andrew K.
Sewell
,
Heather M.
Long
,
Awen M.
Gallimore
,
Pierre
Rizkallah
,
Bruce J.
Maclachlan
,
Andrew
Godkin
Diamond Proposal Number(s):
[20147, 29502, 29990]
Open Access
Abstract: CD4+ T cells recognize a broad range of peptide epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which contribute to immune memory and limit COVID-19 disease. We demonstrate that the immunogenicity of SARS-CoV-2 peptides, in the context of the model allotype HLA-DR1, does not correlate with their binding affinity to the HLA heterodimer. Analyzing six epitopes, some with very low binding affinity, we solve X-ray crystallographic structures of each bound to HLA-DR1. Further structural definitions reveal the precise molecular impact of viral variant mutations on epitope presentation. Omicron escaped ancestral SARS-CoV-2 immunity to two epitopes through two distinct mechanisms: (1) mutations to TCR-facing epitope positions and (2) a mechanism whereby a single amino acid substitution caused a register shift within the HLA binding groove, completely altering the peptide-HLA structure. This HLA-II-specific paradigm of immune escape highlights how CD4+ T cell memory is finely poised at the level of peptide-HLA-II presentation.
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Aug 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Garry
Dolton
,
Cristina
Rius
,
Aaron
Wall
,
Barbara
Szomolay
,
Valentina
Bianchi
,
Sarah A. E.
Galloway
,
Md Samiul
Hasan
,
Théo
Morin
,
Marine E.
Caillaud
,
Hannah L.
Thomas
,
Sarah
Theaker
,
Li Rong
Tan
,
Anna
Fuller
,
Katie
Topley
,
Mateusz
Legut
,
Meriem
Attaf
,
Jade R.
Hopkins
,
Enas
Behiry
,
Joanna
Zabkiewicz
,
Caroline
Alvares
,
Angharad
Lloyd
,
Amber
Rogers
,
Peter
Henley
,
Christopher
Fegan
,
Oliver
Ottmann
,
Stephen
Man
,
Michael D.
Crowther
,
Marco
Donia
,
Inge Marie
Svane
,
David K.
Cole
,
Paul E.
Brown
,
Pierre
Rizkallah
,
Andrew K.
Sewell
Open Access
Abstract: The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such “multipronged” T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.
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Jul 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[20147]
Open Access
Abstract: Tpp80Aa1 from Bacillus thuringiensis is a Toxin_10 family protein (Tpp) with reported action against Culex mosquitoes. Here, we demonstrate an expanded target range, showing Tpp80Aa1 is also active against the larvae of Anopheles gambiae and Aedes aegypti mosquitoes. We report the first crystal structure of Tpp80Aa1 at a resolution of 1.8 Å, which shows Tpp80Aa1 consists of two domains: an N-terminal β-trefoil domain resembling a ricin B lectin and a C-terminal putative pore-forming domain sharing structural similarity with the aerolysin family. Similar to other Tpp family members, we observe Tpp80Aa1 binds to the mosquito midgut, specifically the posterior midgut and the gastric caecum. We also identify that Tpp80Aa1 can interact with galactose-containing glycolipids and galactose, and this interaction is critical for exerting full insecticidal action against mosquito target cell lines.
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Dec 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Garry
Dolton
,
Cristina
Rius
,
Md Samiul
Hasan
,
Aaron
Wall
,
Barbara
Szomolay
,
Enas
Behiry
,
Thomas
Whalley
,
Joel
Southgate
,
Anna
Fuller
,
Théo
Morin
,
Katie
Topley
,
Li Rong
Tan
,
Philip J. R.
Goulder
,
Owen B.
Spiller
,
Pierre J.
Rizkallah
,
Lucy C.
Jones
,
Thomas R.
Connor
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[29502]
Open Access
Abstract: We studied the prevalent cytotoxic CD8 T-cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent Human Leukocyte Antigen (HLA) class I worldwide, HLA A∗02. The Spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘variants of concern’, was not recognised by the large CD8 T-cell response seen across cohorts of HLA A∗02+ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T-cell receptors. Viral escape at prevalent T-cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focussed on a single protein such as SARS-CoV-2 Spike providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T-cell escape in new SARS-CoV-2 variants.
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Jul 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Emily A.
Bates
,
James A.
Davies
,
Jana
Váňová
,
Davor
Nestić
,
Valerie S.
Meniel
,
Sarah
Koushyar
,
Tabitha G.
Cunliffe
,
Rosie M.
Mundy
,
Elise
Moses
,
Hanni K.
Uusi-Kerttula
,
Alexander T.
Baker
,
David K.
Cole
,
Dragomira
Majhen
,
Pierre J.
Rizkallah
,
Toby
Phesse
,
John D.
Chester
,
Alan L.
Parker
Diamond Proposal Number(s):
[18812]
Open Access
Abstract: Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre-existing immunity will be beneficial for future clinical translation. We generated a low seroprevalence HAdV-D10 serotype vector incorporating an αvβ6 integrin selective peptide, A20, to target αvβ6 positive tumour cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation Factor X, potentially eliminating “off-target” hepatic sequestration in vivo. We engineered A20 peptide that selectively binds αvβ6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvβ6+ cancer cell lines, demonstrated significantly increased transduction mediated by αvβ6 targeted variants compared to controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumours. Replication competent HAdV-D10.A20 demonstrated αvβ6 integrin selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions and reduced off-target uptake. Incorporation of an αvβ6 integrin selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation.
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Mar 2022
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