Journal of Molecular Biology
T Cell Receptor Recognition via Cooperative Conformational Plasticity
Introduction
T cell antigen recognition is initiated by molecular contact between the T cell receptor (TCR) and the peptide/major histocompatibility complex (MHC), which leads to an intracellular signaling cascade generally resulting in a functional response by the T cell. Although antigen specificity is considered a hallmark of the adaptive immune response, an increasing body of evidence indicates that the TCR exhibits a degree of cross-reactivity, allowing it to bind and respond to different peptide/MHC ligands.1., 2. Such cross-reactive binding may be important in normal immunological processes such as thymic selection and maintenance of the T cell repertoire, as well as pathogenic conditions such as autoimmunity. Cross-reactivity may be mediated via multiple mechanisms, including molecular mimicry, in which different antigens retain common structural and chemical features,3 or by conformational adjustments within the receptor-ligand interface, whereby structural changes occur in order for binding to proceed. Of these mechanisms, conformational adjustments have received significant attention recently, as the underlying flexibility of the interacting molecules could allow for a broad expansion of available ligands for a given TCR.
Studies performed to date have largely focused on the structural adaptability of the TCR complementarity determining region (CDR)3 loops. Structures of bound and free TCRs have shown that significant changes in CDR3 loop conformation can occur upon binding.4., 5., 6. The role of CDR3 adaptability in TCR cross-reactivity has been confirmed by structures of the same TCR bound to different peptide/MHC complexes showing that CDR3 loops can occupy different conformations depending upon which ligand is bound.7., 8. Less prominent changes in the CDR1 and CDR2 loops have also been seen.5 Small TCR structural changes in regions distal from the CDR loops have also been observed;5., 7. however, there has been no direct evidence that these changes contribute to the ability of a TCR to recognize different ligands.
Conformational adaptability on the peptide/MHC side of the antigen-recognition complex has also been observed. A number of studies have shown that peptides can adopt multiple, distinct conformations in the context of the same MHC molecule,9., 10., 11. whereas others have shown that specific peptide side-chains or even regions of the peptide backbone can be disordered.12., 13., 14., 15., 16. In at least one case, a direct probe of conformational dynamics indicated that MHC polymorphisms can influence peptide mobility.17 Some studies have demonstrated conformational differences in the peptide between the free and TCR-bound complex.7., 18. Yet, little direct evidence exists for conformational changes occurring in a peptide upon TCR binding as an explanatory mechanism for T cell cross-reactivity. Lee et al. found that a native and variant epitope from the HIV gag protein adopted significantly different conformations when bound to the class I MHC HLA-A2.18 Cross-reactive T cells were identified; based on their responses to single amino acid substitutions in the peptides and biophysical binding measurements, the conformation of the two peptides was predicted to be the same after TCR binding. This would suggest that these peptides adopt a common conformation when the TCR binds; however, crystallographic structures of the ternary complexes have not been solved.
Recently, we explored TCR cross-reactivity using as a model system a peptide modified with various hapten groups.19 The haptens were attached to position 5 of the HLA-A2-binding Tax peptide so that they would protrude into the antigen-binding site of the TCR. We were able to generate numerous CD8+ HLA-A2-restricted T cells that could recognize peptides modified with these structurally distinct haptens, although none of the hapten-specific CTL recognized the non-haptenated peptide.
In the current study we extended the analysis of this model system for cross-reactivity using the well-studied αβ TCR A6, which recognizes the Tax 11-19 peptide presented by HLA-A2.20 We examined the response of an A6-bearing T cell clone to the panel of haptenated peptides studied earlier and found that A6 could recognize a derivative of the Tax peptide where tyrosine 5 was replaced with a lysine conjugated to indolyl-butyric acid (Tax-5K-4-(3-Indolyl)-butyric acid; peptide referred to as Tax-5K-IBA; side-chain referred to as Lys-IBA). Although this hapten has an aromatic ring as does the native tyrosine, the presence of a ten atom linker between the position 5 α carbon and the ring, as well as the differences between the substituted phenyl ring of tyrosine and the indole ring of IBA would be expected to impart substantially different chemical and structural features onto the peptide (Figure 1).
We determined that the Tax-5K-IBA peptide acts as a weak agonist for the A6 TCR and that recognition is mediated by a mechanism involving conformational plasticity in both the TCR and the hapten-modified peptide. In the peptide, recognition proceeds with an ordering of the otherwise flexible Lys-IBA side-chain and a shift in the peptide backbone. Conformational changes in the A6 TCR include not only shifts in the CDR3 loops, but also small alterations in Vα/Vβ orientation, which influence the positioning of the TCR constant domains. Recognition of the Tax-5K-IBA ligand in this case thus requires cooperative conformational adjustments on both sides of the TCR/pMHC interface. Our findings illustrate the complex role that protein adaptability or flexibility can play in TCR recognition of ligand, and indicate that cross-reactive TCR-peptide/MHC binding can be achieved through multiple molecular mechanisms, not limited to molecular mimicry and TCR CDR loop shifts alone.
Section snippets
Recognition of a haptenated peptide by a Tax-specific CTL clone
A CTL clone expressing the Tax/HLA-A2-specific A6 TCR was assayed on a panel of haptenated Tax peptides created by substituting the tyrosine at position 5 of the Tax peptide (LLFGYPVYV) with a lysine residue (Tax-5K) to which the various hapten groups were coupled (Supplementary Data, Figure S1). All of these haptenated peptides were able to bind to HLA-A2 and stimulate the generation A2-restricted CD8+ CTL.19 The results demonstrate that the T cell clone RS56, which expresses the A6 TCR,
Discussion
T cell cross-reactivity is a well-accepted phenomenon that is a necessary component of the development and functioning of the normal adaptive immune response.29., 30., 31., 32. Cross-reactive recognition of self-antigens following environmental antigen exposure has also been cited as a potential trigger for certain autoimmune diseases.33., 34., 35., 36., 37. The ability of a T cell to cross-react was originally attributed to molecular mimicry, in which the two antigenic peptides share
Peptides
Haptenated versions of the HLA-A2 binding HTLV-1 Tax11-19 (LLFGYPVYV) peptide62 were synthesized as described.19 All haptens utilized are shown in Supplementary Data, Figure S1 and included DNP, benzoic acid (BENZ), o-iodo-benzoic acid (I-BENZ), oxolinic acid (Oxo), 1-hydroxy-2-naphthoic acid (NA), 3,3-diphenylpropionic acid (DPPA), 2-nitrocinnamic acid (NCA) and 4-(3-indolyl)-butyric acid (IBA). Haptens were purchased from Aldrich (Milwaukee, WI). Peptides were purified by HPLC and dissolved
Acknowledgements
We thank Michael Shiue of Princeton Biomolecules for his technical assistance in peptide design. Funded in part by grant no. GM067079 from NIGMS, NIH (to B.M.B.). O.Y.B. was supported by a fellowship from the Walther Cancer Research Center. GM/CA CAT has been funded in whole or in part with funds from NCI (Y1-CO-1020) and NIGMS (Y1-GM-1104). Use of the Structural Biology Center and GM/CA CAT at the Argonne Advanced Photon Source was supported by the U S Department of Energy under contract no.
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