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]
Open Access
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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Christopher J.
Holland
,
Bruce J.
Maclachlan
,
Valentina
Bianchi
,
Sophie J.
Hesketh
,
Richard
Morgan
,
Owen
Vickery
,
Anna M.
Bulek
,
Anna
Fuller
,
Andrew
Godkin
,
Andrew K.
Sewell
,
Pierre
Rizkallah
,
Stephen
Wells
,
David
Cole
Open Access
Abstract: T-cell immunity is controlled by T cell receptor (TCR) binding to peptide major histocompatibility complexes (pMHCs). The nature of the interaction between these two proteins has been the subject of many investigations because of its central role in immunity against pathogens, cancer, in autoimmunity, and during organ transplant rejection. Crystal structures comparing unbound and pMHC-bound TCRs have revealed flexibility at the interaction interface, particularly from the perspective of the TCR. However, crystal structures represent only a snapshot of protein conformation that could be influenced through biologically irrelevant crystal lattice contacts and other factors. Here, we solved the structures of three unbound TCRs from multiple crystals. Superposition of identical TCR structures from different crystals revealed some conformation differences of up to 5 Å in individual complementarity determining region (CDR) loops that are similar to those that have previously been attributed to antigen engagement. We then used a combination of rigidity analysis and simulations of protein motion to reveal the theoretical potential of TCR CDR loop flexibility in unbound state. These simulations of protein motion support the notion that crystal structures may only offer an artifactual indication of TCR flexibility, influenced by crystallization conditions and crystal packing that is inconsistent with the theoretical potential of intrinsic TCR motions.
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Apr 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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David K.
Cole
,
Anna
Fuller
,
Garry
Dolton
,
Efthalia
Zervoudi
,
Mateusz
Legut
,
Kim
Miles
,
Lori
Blanchfield
,
Florian
Madura
,
Christopher J.
Holland
,
Anna M.
Bulek
,
John S.
Bridgeman
,
John J.
Miles
,
Andrea J. A.
Schauenburg
,
Konrad
Beck
,
Brian D.
Evavold
,
Pierre
Rizkallah
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[4532, 6232]
Abstract: Serial accumulation of mutations to fixation in the SLYNTVATL (SL9) immunodominant, HIV p17 Gag-derived, HLA A2-restricted cytotoxic T lymphocyte epitope produce the SLFNTIAVL triple mutant “ultimate” escape variant. These mutations in solvent-exposed residues are believed to interfere with TCR recognition, although confirmation has awaited structural verification. Here, we solved a TCR co-complex structure with SL9 and the triple escape mutant to determine the mechanism of immune escape in this eminent system. We show that, in contrast to prevailing hypotheses, the main TCR contact residue is 4N and the dominant mechanism of escape is not via lack of TCR engagement. Instead, mutation of solvent-exposed residues in the peptide destabilise the peptide–HLA and reduce peptide density at the cell surface. These results highlight the extraordinary lengths that HIV employs to evade detection by high-affinity TCRs with a broad peptide-binding footprint and necessitate re-evaluation of this exemplar model of HIV TCR escape.
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Nov 2017
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B23-Circular Dichroism
I03-Macromolecular Crystallography
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Amandine
Bovay
,
Vincent
Zoete
,
Garry
Dolton
,
Anna M.
Bulek
,
David K.
Cole
,
Pierre J.
Rizkallah
,
Anna
Fuller
,
Konrad
Beck
,
Olivier
Michielin
,
Daniel E.
Speiser
,
Andrew K.
Sewell
,
Silvia A.
Fuertes Marraco
Diamond Proposal Number(s):
[10462, 10049, 12332]
Abstract: The repertoire of human αβ T cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen-specific T cells and/or exhibit bias towards a TCR gene segment. Here, we studied the TCR repertoire of the HLA-A*0201-restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8+ T cell response to the highly effective YF-17D vaccine. We discover that these A2/LLW-specific CD8+ T cells are highly biased for the TCR α chain TRAV12-2. This bias is already present in A2/LLW-specific naïve T cells before vaccination with YF-17D. Using CD8+ T cell clones, we show that TRAV12-2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline-encoded complementarity determining region (CDR) 1α loop of TRAV12-2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T-cell responses specific for A2/LLW epitope.
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Oct 2017
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B23-Circular Dichroism
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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David K.
Cole
,
Hugo A.
Van Den Berg
,
Angharad
Lloyd
,
Michael D.
Crowther
,
Konrad
Beck
,
Julia
Ekeruche-Makinde
,
John J.
Miles
,
Anna M.
Bulek
,
Garry
Dolton
,
Andrea J.
Schauenburg
,
Aaron
Wall
,
Anna
Fuller
,
Mathew
Clement
,
Bruno
Laugel
,
Pierre J.
Rizkallah
,
Linda
Wooldridge
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[6232, 8096, 10462, 10049, 9308, 12332]
Open Access
Abstract: T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8 T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection.
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Jan 2017
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B23-Circular Dichroism
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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David
Cole
,
Anna
Bulek
,
Garry
Dolton
,
Andrea J.
Schauenberg
,
Barbara
Szomolay
,
William
Rittase
,
Andrew
Trimby
,
Prithiviraj
Jothikumar
,
Anna
Fuller
,
Ania
Skowera
,
Jamie
Rossjohn
,
Cheng
Zhu
,
John
Miles
,
Mark
Peakman
,
Linda
Wooldridge
,
Pierre
Rizkallah
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[7687]
Open Access
Abstract: The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease.
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May 2016
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I04-Macromolecular Crystallography
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David K.
Cole
,
Kim
Miles
,
Florian
Madura
,
Chris
Holland
,
Andrea
Schauenburg
,
Andrew J.
Godkin
,
Anna
Bulek
,
Anna
Fuller
,
Hephzibah
Akpovwa
,
Philip G
Pymm
,
Nathaniel
Liddy
,
Malkit
Sami
,
Yi
Li
,
Pierre
Rizkallah
,
Bent K.
Jakobsen
,
Andrew K.
Sewell
Open Access
Abstract: This work was supported by the Medical Research Council (to A. K. S., T. J. G., and I. B.), Central Laboratory of the Research Councils (CLRC) Daresbury Laboratory, the Diamond Light Source (Midlands BAG MX310), the Danish Medical Research Council (to U. H.), the NOVO Nordic Foundation (to U. H.αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery. ), the Lundbeck Foundation (U. H.), and Fonden til Lægevidenskabens Fremme (to U. H.).
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Jan 2014
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Florian
Madura
,
Pierre J.
Rizkallah
,
Kim M.
Miles
,
Christopher J.
Holland
,
Anna M.
Bulek
,
Anna
Fuller
,
Andrea J. A.
Schauenburg
,
John
Miles
,
Nathaniel
Liddy
,
Malkit
Sami
,
Yi
Li
,
Moushumi
Hossain
,
Brian M.
Baker
,
Bent K.
Jakobsen
,
Andrew K.
Sewell
,
David K.
Cole
Open Access
Abstract: The T-cell receptor (TCR) recognizes peptides bound to major histocompatibility molecules (MHC) and allows T-cells to interrogate the cellular proteome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterized by weak affinity (KD = 100 nm to 270 μm). We used phage-display to produce a melanoma-specific TCR (α24β17) with a 30,000-fold enhanced binding affinity (KD = 0.6 nm) to aid our exploration of the molecular mechanisms utilized to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, α24β17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition.
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May 2013
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: The interaction between the clonotypic αβ T cell receptor (TCR), expressed on the T cell surface, and peptide-major histocompatibility complex (pMHC) molecules, expressed on the target cell surface, governs T cell mediated autoimmunity and immunity against pathogens and cancer. Structural investigations of this interaction have been limited because of the challenges inherent in the production of good quality TCR/pMHC protein crystals. Here, we report the development of an ‘intelligently designed’ crystallization screen that reproducibly generates high quality TCR/pMHC complex crystals suitable for X-ray crystallographic studies, thereby reducing protein consumption. Over the last 2 years, we have implemented this screen to produce 32 T cell related protein structures at high resolution, substantially contributing to the current immune protein database. Protein crystallography, used to study this interaction, has already extended our understanding of the molecular rules that govern T cell immunity. Subsequently, these data may help to guide the intelligent design of T cell based therapies that target human diseases, underlining the importance of developing optimized approaches for crystallizing novel TCR/pMHC complexes.
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Aug 2012
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Anna
Bulek
,
David
Cole
,
Ania
Skowera
,
Garry
Dolton
,
Stephanie
Gras
,
Florian
Madura
,
Anna
Fuller
,
John
Miles
,
Emma
Gostick
,
David A.
Price
,
Jan W.
Drijfhout
,
Robin R.
Knight
,
Guo C.
Huang
,
Nikolai
Lissin
,
Peter E.
Molloy
,
Linda
Fothergill-Gilmore
,
Bent K.
Jakobsen
,
Jamie
Rossjohn
,
Mark
Peakman
,
Pierre J.
Rizkallah
,
Andrew K.
Sewell
Open Access
Abstract: The structural characteristics of the engagement of major histocompatibility complex (MHC) class II–restricted self antigens by autoreactive T cell antigen receptors (TCRs) is established, but how autoimmune TCRs interact with complexes of self peptide and MHC class I has been unclear. Here we examined how CD8+ T cells kill human islet beta cells in type 1 diabetes via recognition of a human leukocyte antigen HLA-A*0201–restricted glucose-sensitive preproinsulin peptide by the autoreactive TCR 1E6. Rigid 'lock-and-key' binding underpinned the 1E6–HLA-A*0201–peptide interaction, whereby 1E6 docked similarly to most MHC class I–restricted TCRs. However, this interaction was extraordinarily weak because of limited contacts with MHC class I. TCR binding was highly peptide centric, dominated by two residues of the complementarity-determining region 3 (CDR3) loops that acted as an 'aromatic-cap' over the complex of peptide and MHC class I (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8+ T cell–mediated autoreactivity.
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Jan 2012
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