Conformation of MHC class II I-Ag7 is sensitive to the P9 anchor amino acid in bound peptide (original) (raw)
Conformation of MHC class II I-Ag7 is sensitive to the
2015
Type I diabetes is a chronic autoimmune disease resulting in the destruction of insulin-producing b cells in the pancreas. In humans, disease incidence is linked to expression of specific MHC class II alleles and in mice type I diabetes is associated with the class II allele I-A g7. I-A g7 contains a polymorphism that is shared by human class II alleles associated with the disease, at position 57 in the b chain, in which aspartic acid is changed to a serine. The P9 pocket in the peptide-binding groove is in part shaped by b57, and therefore the structure of this pocket is modified in I-A g7. Using mAbs, we have previously determined that alternative conformations of I-A g7 form in response to peptide binding. In this study, we have extended these findings by examining how peptides induce I-A g7 molecules to adopt different conformations. By mutating the amino acid in the P9 position of either class II-associated invariant chain peptide (CLIP) or glutamic acid decarboxylase (GAD) 65 (207-220), we have determined that the chemical nature of the P9 anchor amino acid, either acidic or small hydrophobic, affects the overall conformation of the I-A g7 class II molecule. T cell hybridomas specific for GAD 65 (207-220) in the context of I-A g7 were also examined for recognition of I-A g7 bound to GAD 65 (207-220), in which Glu 217 in the P9 position was changed to alanine. We found that although some TCRs were able to recognize both peptides in the context of I-A g7 , and thus both class II conformations, approximately one-third of the T cells tested were not able to recognize the alternate class II conformation formed with the mutated peptide. These results indicate that the I-A g7 conformations may affect functional activation of T cells, and thus may play a role in autoimmunity.
The MHC Class II Molecule I-Ag7 Exists in Alternate Conformations That Are Peptide Dependent
The Journal of Immunology, 2000
Insulin-dependent diabetes mellitus is an autoimmune disease that is genetically linked to the HLA class II molecule DQ in humans and to MHC I-A g7 in nonobese diabetic mice. The I-A g7 -chain is unique and contains multiple polymorphisms, at least one of which is shared with DQ alleles linked to insulin-dependent diabetes mellitus. This polymorphism occurs at position 57 in the -chain, in which aspartic acid is mutated to a serine, a change that results in the loss of an interchain salt bridge between ␣Arg 76 and Asp 57 at the periphery of the peptide binding groove. Using mAbs we have identified alternative conformations of I-A g7 class II molecules. By using an invariant chain construct with various peptides engineered into the class II-associated invariant chain peptide (CLIP) region we have found that formation of these conformations is dependent on the peptide occupying the binding groove. Blocking studies with these Abs indicate that these conformations are present at the cell surface and are capable of interactions with TCRs that result in T cell activation.
The I-Ag7 MHC Class II Molecule Linked to Murine Diabetes Is a Promiscuous Peptide Binder
Journal of Immunology, 2000
Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-A g7 , shares a common ␣-chain with I-A d but has a peculiar -chain. As with most -chain alleles linked to diabetes susceptibility, I-A g7 contains a nonaspartic residue at position 57. We have produced large amounts of empty I-A g7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phagedisplayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-A g7. Structural information was critical in evaluating the binding studies. I-A g7 , like I-A d , appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-A g7 over I-A d as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-A d and I-A g7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the -chain. These characteristics link I-A g7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.
Immunity, 2005
the helical surface )%57-%07ف( is conserved and evolved for biased recognition by the TCR, suggests that TCR/pMHC interactions are tuned for the sampling of different antigens (Daniel et al.). and Structural Biology In support of this hypothesis, examples exist of struc-Stanford University School of Medicine turally diverse, crossreactive peptides to a single T cell Stanford, California 94305 clone (Bhardwaj et al., 1993; Crawford et al., 2004; Hag-2 Torrey Pines Institute for Molecular Studies erty and Allen, 1995; Hemmer et al., 1998a; Ignatowicz San Diego, California 92121 et al., 1997; Kersh and Allen, 1996b; Krogsgaard et al., 2003; Loftus et al., 1999; Reiser et al., 2003; Sykulev et al., 1994; Wucherpfennig and Strominger, 1995). While some of these peptides retain recognizable sequence Summary similarities with the cognate peptides (Crawford et al., 2004; Krogsgaard et al., 2003; Sykulev et al., 1998), other T cell receptor crossreactivity with different peptide crossreactive peptides have been shown to be minimally ligands and biased recognition of MHC are coupled homologous in sequence and therefore presumably enfeatures of antigen recognition that are necessary for gaging the TCR though a unique structural solution the T cell's diverse functional repertoire. In the crystal (Lang et al., 2002; Loftus et al., 1999; Reiser et al., 2003; structure between an autoreactive, EAE T cell clone Wucherpfennig, 2004). 172.10 and myelin basic protein (1-11) presented by In contradiction to the notion of a promiscuous TCR, class II MHC I-A u , recognition of the MHC is dominated most T cell clones are exquisitely sensitive to mutations by the V domain of the TCR, which interacts with the in the peptide (Shih and Allen, 2004). Some of the most MHC ␣ chain in a manner suggestive of a germlineextensively studied TCR/MHC systems, such as 2B4/ encoded TCR/MHC "anchor point." Strikingly, there I-E k (Krogsgaard et al., 2003), 3L.2/I-E k (Kersh et al., are few specific contacts between the TCR CDR3 1998; Shih and Allen, 2004), KRN/I-A g7 (Basu et al., 2000), loops and the MBP peptide. We also find that over and 2C/H-2K b (Degano et al., 2000; Sykulev et al., 1996), 1,000,000 different peptides derived from combinatoexhibit extremely specific peptide recognition and are rial libraries can activate 172.10, yet the TCR strongly largely intolerant of amino acid changes in the TCR conprefers the native MBP contact residues. We suggest tacts. In most reported cases of degenerate TCR recogthat while TCR scanning of pMHC may be degenerate nition, the TCR contact residues of the crossreactive due to the TCR germline bias for MHC, recognition of peptides are similar (Basu et al., 2000; Crawford et al., structurally distinct agonist peptides is not indicative 2004; Grogan et al., 1999; Sykulev et al., 1998; Wilson of TCR promiscuity, but rather highly specific alternaet al., 1999). Indeed, a single centrally located peptide tive solutions to TCR engagement. residue is sufficient to produce tight selectivity by a TCR (Degano et al., 2000; Ding et al., 1998; Krogsgaard et al., Introduction 2003; Shih and Allen, 2004). Thus, the idea of degenerate T cell recognition is difficult to reconcile with experimen-The engagement of the T cell receptor by peptide-MHC tal observations of T cell specificity. is the central antigen-specific event mediating the cellu-To address these questions, we have been studying lar immune response. The concept of an inherent TCR TCR/pMHC interactions in murine experimental allergic degeneracy has emerged to explain how a TCR is able encephalomyelitis (EAE), an intensively studied model to recognize the diverse peptide antigens it encounters system, to understand autoimmunity to neural self-antiduring the processes of thymic education and peripheral gens, such as myelin basic protein (MBP) (Zamvil and surveillance (Ignatowicz et al., 1997; Nikolic-Zugic and Steinman, 1990). The immunodominant encephalito-Bevan, 1990; Hemmer et al., 1998b; Holler and Kranz, genic T cell epitope of MBP, recognized by T cells in 2004; Kersh and Allen, 1996a; Mason, 1998; Wuchermice of the H-2 u haplotype (PL/J or B10.PL), is the acetpfennig, 2004). This concept has been buttressed by ylated N-terminal 11-mer (Ac1-11) (Zamvil et al., 1987). biophysical studies of TCR/MHC interactions (Rudolph The Ac1-11 epitope in the context of class II MHC I-A u and Wilson, 2002), which indicate that flexibility in the has a number of unusual features such as a very short central CDR3 loops of the TCR may serve as an adaptahalf-life (Ͻ15 min.) and a requirement for an N-terminal tion mechanism to "read out" different peptide antigens acetylation, and MBP peptides as short as Ac1-6 can during TCR "scanning" of the universe of peptide-MHC still activate EAE T cell clones (Fairchild et al.Mason et al., The fact that peptide comprises a fraction )%03-%52ف( 1995; Wraith et al., 1992). A crystal structure of I-A u of the composite pMHC surface, while the majority of complexed with MBP1-11 provided a rationale for these properties by finding that the peptide sits in an unusual shifted register in the groove, which results in empty p1 *Correspondence: kcgarcia@stanford.edu 3 These authors contributed equally to this work. and p2 pockets, the MBP N terminus in the p3 pocket, Immunity 82 model included A2-A116 (the ␣ chain of TCR), B3-B117 (the  chain of TCR), C1-C181 (the ␣ chain of I-A u ), D1-D190 (the  chain of I-A u ), Received: August 11, 2004 and P-3-P8 (p-3-P0 is part of the linker and P1-P8 is the MBP Revised: October 7, 2004 peptide). Repeated iterations between manual rebuilding and mini-Accepted: November 17, 2004 mization as well as B factor refinement resulted in a model with R Published: January 25, 2005 factors of 24.3% and R free of 27.4%. The stereochemistry of the structure was analyzed with PROCHECK (Laskowski et al., 1993). References Details of the refinement statistics are given in Supplemental Table S1. Libraries and Peptides Steinman, L. (1988). Limited heterogeneity of T cell receptors from Libraries and biased sublibrary mixtures were prepared at Mixture lymphocytes mediating autoimmune encephalomyelitis allows spe-Sciences, Inc. (San Diego, CA) as described previously (Pinilla et cific immune intervention. Cell 54, 263-273. al., 1994). PCL 97-4 is a synthetic N-acetylated, C-terminal amide, L-amino acid combinatorial decapeptide library arrayed in a posi-Anderton, S.M., Manickasingham, S.P., Burkhart, C., Luckcuck, T.A., Holland, S.J., Lamont, A.G., and Wraith, D.C. (1998). Fine specificity tional scanning format. It consists of 200 mixtures in the OX 9 format, Structure of an Autoimmune TCR/Peptide-MHC Complex 91 of the myelin-reactive T cell repertoire: implications for TCR antago-peptide can induce clinical signs of experimental autoimmune encephalomyelitis. J. Immunol. 161, 60-64. nism in autoimmunity. . Negative selection during the peripheral immune response to antigen. J. Exp. Med. 193, 1-11. Goverman, J. (1999). Tolerance and autoimmunity in TCR transgenic mice specific for myelin basic protein. Immunol. Rev. 169, 147-159. Bankovich, A.J., and Garcia, K.C. (2003). Not just any T cell receptor will do. Immunity 18, 7-11. Goverman, J., Woods, A., Larson, L., Weiner, L.P., Hood, L., and Zaller, D.M. (1993). Transgenic mice that express a myelin basic Basu, D., Horvath, S., Matsumoto, I., Fremont, D.H., and Allen, P.M. protein-specific T cell receptor develop spontaneous autoimmunity. (2000). Molecular basis for recognition of an arthritic peptide and a Cell 72, 551-560. foreign epitope on distinct MHC molecules by a single TCR. software suite for macromolecular structure determination. Acta (2002). Structural snapshot of aberrant antigen presentation linked Crystallogr. D Biol. Crystallogr. 54, 905-921. to autoimmunity: the immunodominant epitope of MBP complexed Buslepp, J., Wang, H., Biddison, W.E., Appella, E., and Collins, E.J. with I-Au. Immunity 17, 83-94. (2003). A correlation between TCR Valpha docking on MHC and CD8 Hemmer, B., Vergelli, M., Gran, B., Ling, N., Conlon, P., Pinilla, C., dependence: implications for T cell selection. Immunity 19, 595-606. Houghten, R., McFarland, H.F., and Martin, R. (1998a). Predictable CCP4 (Collaborative Computational Project, Number 4) (1994). The TCR antigen recognition based on peptide scans leads to the identi-CCP4 suite: programs for protein crystallography. Acta Crystallogr. fication of agonist ligands with no sequence homology. . A basis for alloreactivtions up close. Cell 104, 1-4. ity: MHC helical residues broaden peptide recognition by the TCR. Hennecke, J., Carfi, A., and Wiley, D.C. (2000). Structure of a cova-Immunity 8, 543-552. lently stabilized complex of a human alphabeta T-cell receptor, influ-Davis, M.M., and Bjorkman, P.J. (1988). T-cell antigen receptor enza HA peptide and MHC class II molecule, HLA-DR1. EMBO J. genes and T-cell recognition. Nature 334, 395-402. 19, 5611-5624. Degano, M., Garcia, K.C., Apostolopoulos, V., Rudolph, M.G., Tey-Holler, P.D., and Kranz, D.M. (2004). T cell receptors: affinities, crosston, L., and Wilson, I.A. (2000). A functional hot spot for antigen reactivities, and a conformer model. Mol. Immunol. 40, 1027-1031. recognition in a superagonist TCR/MHC complex. Immunity 12, Houghten, R.A. (1985). General method for the rapid solid-phase 251-261. synthesis of large numbers of peptides: specificity of antigen-anti-Delano, W.L. (2002). The PyMOL Molecular Graphics System (San body interaction at the level of individual amino acids. Proc. Natl. Carlos, CA: DeLano Scientific).