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Distortion of the Major Histocompatibility Complex Class I Binding Groove to Accommodate an Insulin-derived 10-Mer Peptide*

  • Chihiro Motozono
    Footnotes
    Affiliations
    Division of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom

    Department of Immunology, Kinki University School of Medicine, Osaka 589–8511, Japan
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  • James A. Pearson
    Footnotes
    Affiliations
    Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom
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  • Evy De Leenheer
    Affiliations
    Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom
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  • Pierre J. Rizkallah
    Footnotes
    Affiliations
    Division of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom
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  • Konrad Beck
    Affiliations
    Cardiff University School of Dentistry, Heath Park, Cardiff CF14 4XY, United Kingdom
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  • Andrew Trimby
    Affiliations
    Division of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom
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  • Andrew K. Sewell
    Footnotes
    Affiliations
    Division of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom
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  • F. Susan Wong
    Correspondence
    To whom correspondence may be addressed.
    Footnotes
    Affiliations
    Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom
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  • Author Footnotes
    6 A Wellcome Trust Research Career Development Fellow (Grant WT095767).
    David K. Cole
    Correspondence
    To whom correspondence may be addressed.
    Footnotes
    6 A Wellcome Trust Research Career Development Fellow (Grant WT095767).
    Affiliations
    Division of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom
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  • Author Footnotes
    * This work was supported by a Wellcome Trust ISSF grant (to F. S. W., D. K. C., and A. K. S.), United Kingdom Biotechnology and Biological Sciences Research Council Grant BB/H001085/1 (to A. K. S.), and Medical Research Council Grant G0901155 (to F. S. W.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 These authors contributed equally to this work.
    2 Supported by a Diabetes UK PhD studentship.
    3 Supported by a Research Councils United Kingdom fellowship.
    4 A Wellcome Trust Investigator.
    6 A Wellcome Trust Research Career Development Fellow (Grant WT095767).
Open AccessPublished:June 17, 2015DOI:https://doi.org/10.1074/jbc.M114.622522
      The non-obese diabetic mouse model of type 1 diabetes continues to be an important tool for delineating the role of T-cell-mediated destruction of pancreatic β-cells. However, little is known about the molecular mechanisms that enable this disease pathway. We show that insulin reactivity by a CD8+ T-cell clone, known to induce type 1 diabetes, is characterized by weak T-cell antigen receptor binding to a relatively unstable peptide-MHC. The structure of the native 9- and 10-mer insulin epitopes demonstrated that peptide residues 7 and 8 form a prominent solvent-exposed bulge that could potentially be the main focus of T-cell receptor binding. The C terminus of the peptide governed peptide-MHC stability. Unexpectedly, we further demonstrate a novel mode of flexible peptide presentation in which the MHC peptide-binding groove is able to “open the back door” to accommodate extra C-terminal peptide residues.

      Introduction

      Type 1 diabetes (T1D)
      The abbreviations used are: T1D
      type 1 diabetes
      TCR
      T-cell receptor
      pMHCI
      peptide-major histocompatibility complex class I
      NOD
      non-obese diabetic
      Bistris propane
      1,3-bis[tris(hydroxymethyl)methylamino]propane
      HB
      hydrogen bond
      vdW
      van der Waals interaction
      β2m
      β2 microglobulin.
      is an autoimmune disease affecting children and young adults where CD8+ T-cells have recently been shown to play a central role in pancreatic β-cell destruction (
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      ). How these autoreactive CD8+ T-cells escape thymic selection and cause pathology in the periphery is still under debate. However, some evidence suggests that the nature of the interaction between the clonally expressed T-cell receptor (TCR) and self-peptide-major histocompatibility complex class I (pMHCI) may drive this selection. The strength and/or duration of binding between the TCR and pMHCI (
      • Bridgeman J.S.
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      ), can determine the threshold of T-cell activation. Accumulated data suggest that most self-reactive T-cells express TCRs that interact weakly with pMHC compared with pathogenic T-cells (
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      Structural basis for the killing of human beta cells by CD8(+) T cells in type 1 diabetes.
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      ). These observations are compounded by the low stability, predicted or demonstrated, for many autoimmune-pMHC interactions (
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      ). Other molecular investigations of T-cell-induced autoimmunity have also demonstrated suboptimal TCR binding through atypical TCR conformation, compared with most pathogen-specific TCRs (
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      ). These factors have previously been considered to be the basis for poor negative selection of autoreactive T-cells, which may escape from the thymus and become activated in the periphery, potentially through molecular mimicry (
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      ), and thence induce autoimmunity.
      The non-obese diabetic (NOD) mouse model, which develops spontaneous diabetes, has been widely used for investigating T1D (
      • Wong F.S.
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      CD8 T cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells.
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      ,
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      ). There are many parallels between T1D in humans and NOD mice, and findings in these mice have paved the way for important discoveries in humans (
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      ). In NOD mice, in which both genetic susceptibility and environment play a role in disease development, both CD4+ and CD8+ T-cells, recognizing a number of different autoantigens (reviewed in Ref.
      • Babad J.
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      T-cell autoantigens in the non-obese diabetic mouse model of autoimmune diabetes.
      ) are important in autoimmune attack on pancreatic islet β-cells. We have previously cloned a diabetogenic CD8+ T-cell (G9C8) from the islets of young prediabetic NOD mice, which lyses islets in vitro and causes diabetes within 5–10 days after transfer to young non-diabetic NOD mice and NOD.scid mice (
      • Wong F.S.
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      • Wen L.
      • Flavell R.A.
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      ). The G9C8 T-cell clone recognizes insulin B chain amino acids 15–23, and T-cells reacting to this epitope can be highly represented in the small number of cells in the early infiltrate (
      • Wong F.S.
      • Karttunen J.
      • Dumont C.
      • Wen L.
      • Visintin I.
      • Pilip I.M.
      • Shastri N.
      • Pamer E.G.
      • Janeway C.A.
      Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library.
      ), although other specificities become more dominant later. It has been shown that epitopes within the insulin B chain have a prime role in the development of T1D, because substitution at position 16 of the B chain abolishes CD4+ (
      • Nakayama M.
      • Abiru N.
      • Moriyama H.
      • Babaya N.
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      ) and CD8+ T-cell reactivity (
      • Wong F.S.
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      • Dumont C.
      • Wen L.
      • Visintin I.
      • Pilip I.M.
      • Shastri N.
      • Pamer E.G.
      • Janeway C.A.
      Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library.
      ,
      • Wong F.S.
      • Moustakas A.K.
      • Wen L.
      • Papadopoulos G.K.
      • Janeway C.A.
      Analysis of structure and function relationships of an autoantigenic peptide of insulin bound to H-2K(d) that stimulates CD8 T cells in insulin-dependent diabetes mellitus.
      ). This region of the insulin B chain has also been identified as an important autoantigen in humans (
      • Daniel D.
      • Gill R.G.
      • Schloot N.
      • Wegmann D.
      Epitope specificity, cytokine production profile and diabetogenic activity of insulin-specific T cell clones isolated from NOD mice.
      ,
      • Alleva D.G.
      • Crowe P.D.
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      • Kwok W.W.
      • Ling N.
      • Gottschalk M.
      • Conlon P.J.
      • Gottlieb P.A.
      • Putnam A.L.
      • Gaur A.
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      ,
      • Alleva D.G.
      • Gaur A.
      • Jin L.
      • Wegmann D.
      • Gottlieb P.A.
      • Pahuja A.
      • Johnson E.B.
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      ), offering an important model system for investigating the human form of the disease.
      Here, we used cellular and biophysical methods to investigate the molecular interaction between the G9C8 TCR and the native insulin B chain 10-mer peptide, 15LYLVCGERGF24 (G9GF) and 9-mer peptide, 15LYLVCGERG23 (G9G) as well as a heteroclitic form of the peptide, LYLVCGERV (G9V), presented by H-2Kd. G9V was designed to improve MHC stability and has been shown to activate G9C8-like T-cells more strongly than the native G9G peptide (
      • Wong F.S.
      • Moustakas A.K.
      • Wen L.
      • Papadopoulos G.K.
      • Janeway C.A.
      Analysis of structure and function relationships of an autoantigenic peptide of insulin bound to H-2K(d) that stimulates CD8 T cells in insulin-dependent diabetes mellitus.
      ), although the molecular basis for this increased potency has not been fully resolved. We solved the atomic structures of each of the peptides in complex with H-2Kd, demonstrating the peptide residues that interact with the MHC binding groove and identifying the solvent-exposed residues that are most likely to contact the TCR. These data provide the first molecular insight into CD8+ T-cell-induced β-cell destruction via recognition of the insulin B chain in this important disease model of T1D and demonstrate a novel flexible peptide-MHC binding mode that has broad implications for T-cell antigen presentation.

      Discussion

      The NOD mouse model of T1D is an important tool for investigating the role of T-cells in the destruction of islet β-cells in the pancreas. Other molecular investigations of T-cell-induced autoimmunity have shed light on the selection and mode of action of autoreactive T-cells. For example, we have recently demonstrated that a preproinsulin-specific human TCR derived from a CD8+ T-cell bound with extremely weak affinity and a highly focused binding footprint (
      • Bulek A.M.
      • Cole D.K.
      • Skowera A.
      • Dolton G.
      • Gras S.
      • Madura F.
      • Fuller A.
      • Miles J.J.
      • Gostick E.
      • Price D.A.
      • Drijfhout J.W.
      • Knight R.R.
      • Huang G.C.
      • Lissin N.
      • Molloy P.E.
      • Wooldridge L.
      • Jakobsen B.K.
      • Rossjohn J.
      • Peakman M.
      • Rizkallah P.J.
      • Sewell A.K.
      Structural basis for the killing of human beta cells by CD8(+) T cells in type 1 diabetes.
      ). Other studies of autoreactive T-cells in other disease models have also demonstrated suboptimal TCR binding, either through weak TCR affinity (
      • Hahn M.
      • Nicholson M.J.
      • Pyrdol J.
      • Wucherpfennig K.W.
      Unconventional topology of self peptide-major histocompatibility complex binding by a human autoimmune T cell receptor.
      ), poor pMHC stability (
      • Yin Y.
      • Li Y.
      • Kerzic M.C.
      • Martin R.
      • Mariuzza R.A.
      Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection.
      ), topologically unusual TCR binding (
      • Sethi D.K.
      • Schubert D.A.
      • Anders A.-K.
      • Heroux A.
      • Bonsor D.A.
      • Thomas C.P.
      • Sundberg E.J.
      • Pyrdol J.
      • Wucherpfennig K.W.
      A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC.
      ,
      • Hahn M.
      • Nicholson M.J.
      • Pyrdol J.
      • Wucherpfennig K.W.
      Unconventional topology of self peptide-major histocompatibility complex binding by a human autoimmune T cell receptor.
      ), or a combination (
      • Bulek A.M.
      • Cole D.K.
      • Skowera A.
      • Dolton G.
      • Gras S.
      • Madura F.
      • Fuller A.
      • Miles J.J.
      • Gostick E.
      • Price D.A.
      • Drijfhout J.W.
      • Knight R.R.
      • Huang G.C.
      • Lissin N.
      • Molloy P.E.
      • Wooldridge L.
      • Jakobsen B.K.
      • Rossjohn J.
      • Peakman M.
      • Rizkallah P.J.
      • Sewell A.K.
      Structural basis for the killing of human beta cells by CD8(+) T cells in type 1 diabetes.
      ,
      • Hahn M.
      • Nicholson M.J.
      • Pyrdol J.
      • Wucherpfennig K.W.
      Unconventional topology of self peptide-major histocompatibility complex binding by a human autoimmune T cell receptor.
      ). These observations have led to the suggestion that autoreactive T-cells receive weak or unconventional signals in the thymus that lead to positive selection rather than deletion. Here, we show that the G9C8 T-cell clone is reactive to an autoantigenic peptide that is part of the insulin protein but that the native epitopes were both relatively unstable compared with a heteroclitic peptide with optimal anchor residues. The TCR from this clone bound with weak affinity to the native epitopes, resulting in lower functional avidity. This combination adds support to the notion that selection of this clone could occur through weak T-cell signaling in the thymus. T-cells that have high affinity TCRs for more stable insulin-derived epitopes would probably be deleted through negative selection, explaining their absence in the periphery. The high levels of insulin expressed by β-cells and the probable high levels of G9G/G9GF epitopes on the surface of these cells might bridge the activation threshold of G9C8-like T-cells, inducing the autoreactivity observed. Surprisingly, despite a weak monomeric affinity for the G9C8 TCR, H-2Kd·G9G tetramers could still robustly identify cognate T-cells. It is possible that the comparatively strong murine pMHC-CD8 affinity, compared with human pMHC-CD8 (
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      ,
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      • Bell J.I.
      • Sewell A.K.
      The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor ζ chain.
      ), could play a role in stabilizing this weak affinity interaction at the cell surface (
      • Wooldridge L.
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      • Glick M.
      • Gostick E.
      • Laugel B.
      • Hutchinson S.L.
      • Milicic A.
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      • Price D.A.
      • Sewell A.K.
      Interaction between the CD8 coreceptor and major histocompatibility complex class I stabilizes T cell receptor-antigen complexes at the cell surface.
      ,
      • Melenhorst J.J.
      • Scheinberg P.
      • Chattopadhyay P.K.
      • Lissina A.
      • Gostick E.
      • Cole D.K.
      • Wooldridge L.
      • van den Berg H.A.
      • Bornstein E.
      • Hensel N.F.
      • Douek D.C.
      • Roederer M.
      • Sewell A.K.
      • Barrett A.J.
      • Price D.A.
      Detection of low avidity CD8+ T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers.
      ). Importantly, the G9C8 T-cell did not express an inherently weak binding TCR, because peptide substitution of Gly to Val at position 9 resulted in anti-viral-like affinity (
      • Bridgeman J.S.
      • Sewell A.K.
      • Miles J.J.
      • Price D.A.
      • Cole D.K.
      Structural and biophysical determinants of αβ T-cell antigen recognition.
      ,
      • Cole D.K.
      • Pumphrey N.J.
      • Boulter J.M.
      • Sami M.
      • Bell J.I.
      • Gostick E.
      • Price D.A.
      • Gao G.F.
      • Sewell A.K.
      • Jakobsen B.K.
      Human TCR-binding affinity is governed by MHC class restriction.
      ,
      • Aleksic M.
      • Liddy N.
      • Molloy P.E.
      • Pumphrey N.
      • Vuidepot A.
      • Chang K.-M.
      • Jakobsen B.K.
      Different affinity windows for virus and cancer-specific T-cell receptors: implications for therapeutic strategies.
      ). The corresponding enhanced tetramer staining using the G9V peptide paves the way for the development of improved reagents to isolate, phenotype, and clonotype insulin-reactive CD8+ T-cells to better follow and determine their role in disease progression. Furthermore, this demonstration that the G9C8 TCR could bind to an altered ligand with >10 times higher affinity compared with the native ligands opens up the intriguing possibility that this T-cell clone could potentially be primed by a more immunogenic target and then cross-react with insulin B chain epitopes expressed by β-cells through a molecular mimicry type mechanism. Thus, this altered ligand could also be used to test the potential role of molecular mimicry on disease outcome.
      The stability of the pMHC complex is critical in the presentation of epitopes to T-cells, because unstable pMHC will be present at lower concentrations or absent on the surface of antigen-presenting cells. To confound this issue, previously (
      • Cole D.K.
      • Edwards E.S.J.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ,
      • Cole D.K.
      • Yuan F.
      • Rizkallah P.J.
      • Miles J.J.
      • Gostick E.
      • Price D.A.
      • Gao G.F.
      • Jakobsen B.K.
      • Sewell A.K.
      Germ line-governed recognition of a cancer epitope by an immunodominant human T-cell receptor.
      ,
      • Uchtenhagen H.
      • Abualrous E.T.
      • Stahl E.
      • Allerbring E.B.
      • Sluijter M.
      • Zacharias M.
      • Sandalova T.
      • van Hall T.
      • Springer S.
      • Nygren P.-Å.
      • Achour A.
      Proline substitution independently enhances H-2D(b) complex stabilization and TCR recognition of melanoma-associated peptides.
      ) and here, we found that modifications that altered pMHC stability also had a large effect on TCR binding affinity. H-2Kd is unusual compared with the binding motif for most other mouse alleles (that have an anchor at position 5 and the C terminus) in that it anchors at positions 2 and the C terminus, reminiscent of most human peptide-MHC binding motifs. Thus, our observations concerning the effects of TCR binding affinity upon altering the C-terminal anchor could be unique to this murine MHC allele. Because the peptide modifications were located at the C terminus, and the most solvent exposed peptide residues were Glu-7 and Arg-8, it is reasonable to speculate that the G9C8 TCR focuses on the C terminus of the peptide. This is consistent with our previous data demonstrating that modification of these residues (particularly Arg-8), along with Val-4, which was also pointing out of the groove according to our structural analysis, reduced T-cell activation (
      • Wong F.S.
      • Moustakas A.K.
      • Wen L.
      • Papadopoulos G.K.
      • Janeway C.A.
      Analysis of structure and function relationships of an autoantigenic peptide of insulin bound to H-2K(d) that stimulates CD8 T cells in insulin-dependent diabetes mellitus.
      ,
      • Petrich de Marquesini L.G.
      • Moustakas A.K.
      • Thomas I.J.
      • Wen L.
      • Papadopoulos G.K.
      • Wong F.S.
      Functional inhibition related to structure of a highly potent insulin-specific CD8 T cell clone using altered peptide ligands.
      ). This observation could explain the strong binding affinity between the G9C8 TCR and G9V, because Val-9 might stabilize the main TCR-peptide contact region, enabling more optimal contacts. Binding to this region of the peptide may also explain why, although the G9C8 TCR bound with a similarly weak affinity to H-2Kd·G9G and H-2Kd·G9GF, the G9C8 T-cell was more sensitive to the G9G peptide. The unusual presentation mode of the G9GF peptide may affect the dynamics of TCR binding, perhaps altering the formation of an optimal immune synapse or inhibiting the formation of TCR catch bonds that have recently been shown to play an important role in T-cell activation (
      • Liu B.
      • Chen W.
      • Evavold B.D.
      • Zhu C.
      Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling.
      ).
      Our structural investigations also revealed a novel and unexpected mode of peptide presentation that has far reaching implications for T-cell antigen recognition in general. Although structures of different length versions of the same peptide have been published before, this is the first example in which the peptide alters the shape of the MHCI binding groove to accommodate an extra residue in the F-pocket. Additional residues at the N terminus and C terminus have been shown to have the following effects: 1) the central bulge of the peptide was altered because the extra residue could not be accommodated by the closed N-terminal end of the MHC binding groove (
      • Tynan F.E.
      • Borg N.A.
      • Miles J.J.
      • Beddoe T.
      • El-Hassen D.
      • Silins S.L.
      • van Zuylen W.J.M.
      • Purcell A.W.
      • Kjer-Nielsen L.
      • McCluskey J.
      • Burrows S.R.
      • Rossjohn J.
      High resolution structures of highly bulged viral epitopes bound to major histocompatibility complex class I. Implications for T-cell receptor engagement and T-cell immunodominance.
      ,
      • Tynan F.E.
      • Burrows S.R.
      • Buckle A.M.
      • Clements C.S.
      • Borg N.A.
      • Miles J.J.
      • Beddoe T.
      • Whisstock J.C.
      • Wilce M.C.
      • Silins S.L.
      • Burrows J.M.
      • Kjer-Nielsen L.
      • Kostenko L.
      • Purcell A.W.
      • McCluskey J.
      • Rossjohn J.
      T cell receptor recognition of a “super-bulged” major histocompatibility complex class I-bound peptide.
      • Ekeruche-Makinde J.
      • Miles J.J.
      • van den Berg H.A.
      • Skowera A.
      • Cole D.K.
      • Dolton G.
      • Schauenburg A.J.A.
      • Tan M.P.
      • Pentier J.M.
      • Llewellyn-Lacey S.
      • Miles K.M.
      • Bulek A.M.
      • Clement M.
      • Williams T.
      • Trimby A.
      • Bailey M.
      • Rizkallah P.
      • Rossjohn J.
      • Peakman M.
      • Price D.A.
      • Burrows S.R.
      • Sewell A.K.
      • Wooldridge L.
      Peptide length determines the outcome of TCR/peptide-MHCI engagement.
      ); 2) the 9-mer version of the peptide assumed the same conformation as the 10-mer version of the peptide by using peptide residue 1, rather than residue 2, as the anchor (
      • Borbulevych O.Y.
      • Insaidoo F.K.
      • Baxter T.K.
      • Powell Jr., D.J.
      • Johnson L.A.
      • Restifo N.P.
      • Baker B.M.
      Structures of MART-126/27–35 peptide/HLA-A2 complexes reveal a remarkable disconnect between antigen structural homology and T cell recognition.
      ); or 3) extra residues protruded from the groove at the peptide termini (
      • Collins E.J.
      • Garboczi D.N.
      • Wiley D.C.
      Three-dimensional structure of a peptide extending from one end of a class I MHC binding site.
      ,
      • Tenzer S.
      • Wee E.
      • Burgevin A.
      • Stewart-Jones G.
      • Friis L.
      • Lamberth K.
      • Chang C.H.
      • Harndahl M.
      • Weimershaus M.
      • Gerstoft J.
      • Akkad N.
      • Klenerman P.
      • Fugger L.
      • Jones E.Y.
      • McMichael A.J.
      • Buus S.
      • Schild H.
      • van Endert P.
      • Iversen A.K.N.
      Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance.
      ). Here, the C-terminal end of the 10-mer G9GF peptide formed a dynamic interaction with the MHC binding groove. H-2Kd·G9GF crystallized with three molecules in the asymmetric unit, demonstrating two distinct conformations. The dominant conformation observed in two of the copies (G9GF-stretched) forced the MHC binding groove to open to accommodate the bulky side chain of Phe-10, resulting in MHC residue Tyr-84 swinging 8.2 Å and altering the shape of the MHC F-pocket. Usually, the central residues of longer peptides are squeezed into more extended conformations because of the closed nature of the MHCI binding groove, as observed in the G9GF-bulged model of the structure. In the G9GF-stretched structure, the movement around the MHC F-pocket enabled the C terminus of the G9GF peptide to slide further down the groove so that the N terminus of the peptide could adopt a potentially similar conformation to the G9G and G9V 9-mer peptides. The ability of the G9GF 10-mer peptide to “mimic” the conformation of the 9-mer peptides is likely to be an important factor facilitating recognition of the G9GF peptide by the G9C8 TCR. The dynamic nature of the MHC binding groove was highly unexpected and adds to other studies in which a distinct movement in the MHC helices and/or peptide has been observed (
      • Borbulevych O.Y.
      • Piepenbrink K.H.
      • Gloor B.E.
      • Scott D.R.
      • Sommese R.F.
      • Cole D.K.
      • Sewell A.K.
      • Baker B.M.
      T cell receptor cross-reactivity directed by antigen-dependent tuning of peptide-MHC molecular flexibility.
      ,
      • Tynan F.E.
      • Reid H.H.
      • Kjer-Nielsen L.
      • Miles J.J.
      • Wilce M.C.J.
      • Kostenko L.
      • Borg N.A.
      • Williamson N.A.
      • Beddoe T.
      • Purcell A.W.
      • Burrows S.R.
      • McCluskey J.
      • Rossjohn J.
      A T cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class I molecule.
      ). Combined, these data provide important evidence demonstrating the highly flexible nature of peptide presentation by MHC. Interestingly, a previous study implemented mutation of Arg-84 for Ala-84 for the stable generation of a single chain pMHC (
      • Lybarger L.
      • Yu Y.Y.L.
      • Miley M.J.
      • Fremont D.H.
      • Myers N.
      • Primeau T.
      • Truscott S.M.
      • Connolly J.M.
      • Hansen T.H.
      Enhanced immune presentation of a single-chain major histocompatibility complex class I molecule engineered to optimize linkage of a C-terminally extended peptide.
      ). Our findings would suggest that this mutation could have a substantial effect on the shape and dynamics of the MHC F pocket, leading to potential changes in peptide presentation. The flexibility we observed around the F-pocket also has implications for so-called TCR-pMHC “catch bonds.” A recent study demonstrated that, under force, some TCR and pMHC interactions can become stronger, resulting in enhanced T-cell activation (
      • Liu B.
      • Chen W.
      • Evavold B.D.
      • Zhu C.
      Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling.
      ). The formation of catch bonds suggests that the TCR, pMHC, or both undergo structural rearrangements when under force during binding at the cell surface, explaining the increase in binding strength. Our data, demonstrating the potential dynamic nature of the region around the MHC F-pocket, fits well with the notion of catch bond formation.
      In summary, we show that insulin reactivity by a CD8+ T-cell clone, known to induce T1D, is characterized by weak TCR affinity to a highly unstable pMHC. The G9C8 TCR was able to bind more strongly to a peptide altered at the C terminus, demonstrating the potential of this T-cell clone to be triggered by a more immunogenic target. This observation also suggests that the interaction between the TCR and pMHC is likely to be focused toward the C terminus of the peptide, explaining the difference in sensitivity between the C-terminally altered peptide ligands investigated. Finally, we demonstrate a novel mode of flexible peptide presentation in which the MHC can effectively “open the back door” to accommodate extra C-terminal peptide residues.

      Author Contributions

      C. M., J. A. P., E. D. L., P. J. R., K. B., A. T., and D. K. C. performed experiments. P. J. R. and D. K. C. performed the structural analysis. A. K. S., F. S. W., and D. K. C. conceived and funded the study and wrote the manuscript.

      Acknowledgments

      We thank Professor David Margulies and Professor George Papadopoulos for critical reading of the manuscript. We also thank the staff at Diamond Light Source for providing facilities and support.

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