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Open Access
Abstract: The CD8 T cell response to the HLA-A2-restricted epitope LLWNGPMAV (LLW) of the non-structural protein 4b of Yellow Fever Virus (YFV) is remarkably immunodominant, highly prevalent and powerful in YFV-vaccinated humans. Here we used a combinatorial peptide library screening in the context of an A2/LLW-specific CD8 T cell clone to identify a superagonist that features a methionine to isoleucine substitution at position 7. Based on in silico modeling, the functional enhancement of this LLW-7I mutation was associated with alterations in the structural dynamics of the peptide in the major histocompatibility complex (pMHC) binding with the T cell receptor (TCR). While the TCR off-rate of LLW-7I pMHC is comparable to the wild type peptide, the rigidity of the 7I peptide seems to confer less entropy loss upon TCR binding. This LLW-7I superagonist is an example of improved functionality in human CD8 T cells associated with optimized ligand rigidity for TCR binding and not with changes in TCR:pMHC off-rate kinetics.
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Sep 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Christopher J.
Holland
,
Rory M.
Crean
,
Johanne M.
Pentier
,
Ben
De Wet
,
Angharad
Lloyd
,
Velupillai
Srikannathasan
,
Nikolai
Lissin
,
Katy A.
Lloyd
,
Thomas H.
Blicher
,
Paul J.
Conroy
,
Miriam
Hock
,
Robert J.
Pengelly
,
Thomas E.
Spinner
,
Brian
Cameron
,
Elizabeth A.
Potter
,
Anitha
Jeyanthan
,
Peter E.
Molloy
,
Malkit
Sami
,
Milos
Aleksic
,
Nathaniel
Liddy
,
Ross A.
Robinson
,
Stephen
Harper
,
Marco
Lepore
,
Chris R.
Pudney
,
Marc W.
Van Der Kamp
,
Pierre J.
Rizkallah
,
Bent K.
Jakobsen
,
Annelise
Vuidepot
,
David K.
Cole
Diamond Proposal Number(s):
[17077, 14843]
Abstract: Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.
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Apr 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14843]
Abstract: Calpain is a Ca2+-activated, heterodimeric cysteine protease consisting of a large catalytic subunit and a small regulatory subunit. Dysregulation of this enzyme is involved in a range of pathological conditions such as cancer, Alzheimer's disease and rheumatoid arthritis, and thus calpain I is a drug target with potential therapeutic applications. Difficulty in the production of this enzyme has hindered structural and functional investigations in the past, although heterodimeric calpain I can be generated by Escherichia coli expression in low yield. Here, an unexpected structure discovered during crystallization trials of heterodimeric calpain I (CAPN1C115S + CAPNS1ΔGR) is reported. A novel co-crystal structure of the PEF(S) domain from the dissociated regulatory small subunit of calpain I and the RNA-binding chaperone Hfq, which was likely to be overproduced as a stress response to the recombinant expression conditions, was obtained, providing unexpected insight in the chaperone function of Hfq.
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Feb 2020
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I02-Macromolecular Crystallography
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Bruce J.
Maclachlan
,
Garry
Dolton
,
Athanasios
Papakyriakou
,
Alexander
Greenshields-watson
,
Georgina H.
Mason
,
Andrea
Schauenburg
,
Matthieu
Besneux
,
Barbara
Szomolay
,
Tim
Elliott
,
Andrew K.
Sewell
,
Awen
Gallimore
,
Pierre
Rizkallah
,
David K.
Cole
,
Andrew
Godkin
Diamond Proposal Number(s):
[10462]
Abstract: CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide flanking residues (PFRs) can extend beyond the limits of the HLA-binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence TCR affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak T-cell receptor (TCR) binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111-130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak-affinity TCR-pHLA-II interactions.
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Oct 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14843]
Open Access
Abstract: Adenoviruses are clinically important agents. They cause respiratory distress, gastroenteritis, and epidemic keratoconjunctivitis. As non-enveloped, double-stranded DNA viruses, they are easily manipulated, making them popular vectors for therapeutic applications, including vaccines. Species D adenovirus type 26 (HAdV-D26) is both a cause of EKC and other diseases and a promising vaccine vector. HAdV-D26–derived vaccines are under investigation as protective platforms against HIV, Zika, and respiratory syncytial virus infections and are in phase 3 clinical trials for Ebola. We recently demonstrated that HAdV-D26 does not use CD46 or Desmoglein-2 as entry receptors, while the putative interaction with coxsackie and adenovirus receptor is low affinity and unlikely to represent the primary cell receptor. Here, we establish sialic acid as a primary entry receptor used by HAdV-D26. We demonstrate that removal of cell surface sialic acid inhibits HAdV-D26 infection, and provide a high-resolution crystal structure of HAdV-D26 fiber-knob in complex with sialic acid.
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Sep 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Harley L.
Worthy
,
Husam Sabah
Auhim
,
W. David
Jamieson
,
Jacob R.
Pope
,
Aaron
Wall
,
Robert
Batchelor
,
Rachel L.
Johnson
,
Daniel W.
Watkins
,
Pierre
Rizkallah
,
Oliver K.
Castell
,
D. Dafydd
Jones
Diamond Proposal Number(s):
[14843]
Open Access
Abstract: Construction of artificial higher order protein complexes allows sampling of structural architectures and functional features not accessible by classical monomeric proteins. Here, we combine in silico modelling with expanded genetic code facilitated strain promoted azide-alkyne cycloaddition to construct artificial complexes that are structurally integrated protein dimers and demonstrate functional synergy. Using fluorescent proteins sfGFP and Venus as models, homodimers and heterodimers are constructed that switched ON once assembled and display enhanced spectral properties. Symmetrical crosslinks are found to be important for functional enhancement. The determined molecular structure of one artificial dimer shows that a new long-range polar network comprised mostly of organised water molecules links the two chromophores leading to activation and functional enhancement. Single molecule analysis reveals the dimer is more resistant to photobleaching spending longer times in the ON state. Thus, genetically encoded bioorthogonal chemistry can be used to generate truly integrated artificial protein complexes that enhance function.
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Jul 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14843]
Abstract: Light-oxygen-voltage (LOV) domains are increasingly used to engineer photoresponsive biological systems. While the photochemical cycle is well documented, the allosteric mechanism by which formation of a cysteinyl-flavin adduct leads to activation is unclear. Via replacement of flavin mononucleotide (FMN) with 5-deazaflavin mononucleotide (5dFMN) in the Aureochrome1a (Au1a) transcription factor from Ochromonas danica, a thermally stable cysteinyl-5dFMN adduct was generated. High-resolution crystal structures (<2 Å) under different illumination conditions with either FMN or 5dFMN chromophores reveal three conformations of the highly conserved glutamine 293. An allosteric hydrogen bond network linking the chromophore via Gln293 to the auxiliary A′α helix is observed. With FMN, a “flip” of the Gln293 side chain occurs between dark and lit states. 5dFMN cannot hydrogen bond through the C5 position and proved to be unable to support Au1a domain dimerization. Under blue light, the Gln293 side chain instead “swings” away in a conformation distal to the chromophore and not previously observed in existing LOV domain structures. Together, the multiple side chain conformations of Gln293 and functional analysis of 5dFMN provide new insight into the structural requirements for LOV domain activation.
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May 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[1484]
Abstract: Many aldehydes such as furfural are present in high quantities in lignocellulose lysates and are fermentation inhibitors that make biofuel production from this abundant carbon source extremely challenging. Cbei_3974 has recently been identified as an aldo-keto reductase responsible for partial furfural resistance in Clostridium beijerinkii. Rational engineering of this enzyme could enhance the furfural tolerance of this organism thereby improving biofuel yields. We report an extensive characterization of Cbei_3974 and a single crystal X-ray structure of Cbei_3974 in complex with NADPH at a resolution of 1.75 Å. Docking studies identified residues involved in substrate binding and an activity screen revealed the substrate tolerance of the enzyme. Hydride transfer, which is partially rate limiting under physiological conditions, occurs from the pro-R hydrogen of NADPH. Enzyme isotope labeling revealed a temperature-independent enzyme isotope effect of unity, indicating that the enzyme does not use dynamic coupling for catalysis and suggests that the active site of the enzyme is optimally configured for catalysis with the substrate tested.
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May 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Florian
Madura
,
Pierre J.
Rizkallah
,
Mateusz
Legut
,
Christopher J.
Holland
,
Anna
Fuller
,
Anna
Bulek
,
Andrea J.
Schauenburg
,
Andrew
Trimby
,
Jade R.
Hopkins
,
Stephen
Wells
,
Andrew
Godkin
,
John J.
Miles
,
Malkit
Sami
,
Yi
Li
,
Nathaniel
Liddy
,
Bent K.
Jakobsen
,
E. Joel
Loveridge
,
David K.
Cole
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[4352, 6232]
Abstract: The HLA‐A*02:01‐restricted decapeptide EAAGIGILTV, derived from Melanoma Antigen Recognized by T‐cells‐1 (MART‐1) protein, represents one of the best‐studied tumor associated T‐cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptides are presented in distinct conformations by HLA‐A*02:01 and TCRs from clinically relevant T‐cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5‐HLA‐A*02:01‐AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide‐MHC anchoring. This ‘flexing’ at the TCR‐peptide‐MHC interface to accommodate the peptide antigen explains previously‐observed incongruences in this well‐studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T‐cells.
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May 2019
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I24-Microfocus Macromolecular Crystallography
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Sarah A. E.
Galloway
,
Garry
Dolton
,
Meriem
Attaf
,
Aaron
Wall
,
Anna
Fuller
,
Cristina
Rius
,
Valentina
Bianchi
,
Sarah
Theaker
,
Angharad
Lloyd
,
Marine E.
Caillaud
,
Inge Marie
Svane
,
Marco
Donia
,
David K.
Cole
,
Barbara
Szomolay
,
Pierre
Rizkallah
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[14843]
Open Access
Abstract: Recent immunotherapeutic approaches using adoptive cell therapy, or checkpoint blockade, have demonstrated the powerful anti-cancer potential of CD8 cytotoxic T-lymphocytes (CTL). While these approaches have shown great promise, they are only effective in some patients with some cancers. The potential power, and relative ease, of therapeutic vaccination against tumour associated antigens (TAA) present in different cancers has been a long sought-after approach for harnessing the discriminating sensitivity of CTL to treat cancer and has seen recent renewed interest following cancer vaccination successes using unique tumour neoantigens. Unfortunately, results with TAA-targeted “universal” cancer vaccines (UCV) have been largely disappointing. Infectious disease models have demonstrated that T-cell clonotypes that recognise the same antigen should not be viewed as being equally effective. Extrapolation of this notion to UCV would suggest that the quality of response in terms of the T-cell receptor (TCR) clonotypes induced might be more important than the quantity of the response. Unfortunately, there is little opportunity to assess the effectiveness of individual T-cell clonotypes in vivo. Here, we identified effective, persistent T-cell clonotypes in an HLA A2+ patient following successful tumour infiltrating lymphocyte (TIL) therapy. One such T-cell clone was used to generate super-agonist altered peptide ligands (APLs). Further refinement produced an APL that was capable of inducing T-cells in greater magnitude, and with improved effectiveness, from the blood of all 14 healthy donors tested. Importantly, this APL also induced T-cells from melanoma patient blood that exhibited superior recognition of the patient's own tumour compared to those induced by the natural antigen sequence. These results suggest that use of APL to skew the clonotypic quality of T-cells induced by cancer vaccination could provide a promising avenue in the hunt for the UCV “magic bullet.”
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Mar 2019
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