The Hypervariable Loops of Free TCRs Sample Multiple Distinct Metastable Conformations in Solution.
ABSTRACT: CD4+ and CD8+ ?? T cell antigen recognition is determined by the interaction between the TCR Complementarity Determining Region (CDR) loops and the peptide-presenting MHC complex. These T cells are known for their ability to recognize multiple pMHC complexes, and for a necessary promiscuity that is required for their selection and function in the periphery. Crystallographic studies have previously elucidated the role of structural interactions in TCR engagement, but our understanding of the dynamic process that occurs during TCR binding is limited. To better understand the dynamic states that exist for TCR CDR loops in solution, and how this relates to their states when in complex with pMHC, we simulated the 2C T cell receptor in solution using all-atom molecular dynamics in explicit water and constructed a Markov State Model for each of the CDR3? and CDR3? loops. These models reveal multiple metastable states for the CDR3 loops in solution. Simulation data and metastable states reproduce known CDR3? crystal conformations, and reveal several novel conformations suggesting that CDR3? bound states are the result of search processes from nearby pre-existing equilibrium conformational states. Similar simulations of the invariant, Type I Natural Killer T cell receptor NKT15, which engages the monomorphic, MHC-like CD1d ligand, demonstrate that iNKT TCRs also have distinct states, but comparatively restricted degrees of motion.
Project description:?? 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.
Project description:TCRep 3D is an automated systematic approach for TCR-peptide-MHC class I structure prediction, based on homology and ab initio modeling. It has been considerably generalized from former studies to be applicable to large repertoires of TCR. First, the location of the complementary determining regions of the target sequences are automatically identified by a sequence alignment strategy against a database of TCR V? and V? chains. A structure-based alignment ensures automated identification of CDR3 loops. The CDR are then modeled in the environment of the complex, in an ab initio approach based on a simulated annealing protocol. During this step, dihedral restraints are applied to drive the CDR1 and CDR2 loops towards their canonical conformations, described by Al-Lazikani et. al. We developed a new automated algorithm that determines additional restraints to iteratively converge towards TCR conformations making frequent hydrogen bonds with the pMHC. We demonstrated that our approach outperforms popular scoring methods (Anolea, Dope and Modeller) in predicting relevant CDR conformations. Finally, this modeling approach has been successfully applied to experimentally determined sequences of TCR that recognize the NY-ESO-1 cancer testis antigen. This analysis revealed a mechanism of selection of TCR through the presence of a single conserved amino acid in all CDR3? sequences. The important structural modifications predicted in silico and the associated dramatic loss of experimental binding affinity upon mutation of this amino acid show the good correspondence between the predicted structures and their biological activities. To our knowledge, this is the first systematic approach that was developed for large TCR repertoire structural modeling.
Project description:Although conformational changes in TCRs and peptide Ags presented by MHC protein (pMHC) molecules often occur upon binding, their relationship to intrinsic flexibility and role in ligand selectivity are poorly understood. In this study, we used nuclear magnetic resonance to study TCR-pMHC binding, examining recognition of the QL9/H-2L(d) complex by the 2C TCR. Although the majority of the CDR loops of the 2C TCR rigidify upon binding, the CDR3? loop remains mobile within the TCR-pMHC interface. Remarkably, the region of the QL9 peptide that interfaces with CDR3? is also mobile in the free pMHC and in the TCR-pMHC complex. Determination of conformational exchange kinetics revealed that the motions of CDR3? and QL9 are closely matched. The matching of conformational exchange in the free proteins and its persistence in the complex enhances the thermodynamic and kinetic stability of the TCR-pMHC complex and provides a mechanism for facile binding. We thus propose that matching of structural fluctuations is a component of how TCRs scan among potential ligands for those that can bind with sufficient stability to enable T cell signaling.
Project description:The conserved diagonal docking mode observed in structures of T-cell receptors (TCRs) bound to peptide-MHC ligands is believed to reflect coevolution of TCR and MHC genes. This coevolution is supported by the conservation of certain interactions between the germ-line-encoded complementarity-determining region (CDR)1 and CDR2 loops of TCR and MHC. However, the rules governing these interactions are not straightforward, even when the same variable (V) region recognizes the same MHC molecule. Here, we demonstrate that the somatically generated CDR3 loops can markedly alter evolutionarily selected contacts between TCR and MHC ("CDR3 editing"). To understand CDR3 editing at the atomic level, we determined the structure of a human melanoma-specific TCR (G4) bound to the MHC class II molecule HLA-DR1 and an epitope from mutant triose phosphate isomerase (mutTPI). A comparison of the G4-mutTPI-DR1 complex with a complex involving a TCR (E8) that uses the same V? region to recognize the same mutTPI-DR1 ligand as G4 revealed that CDR1? adopts markedly different conformations in the two TCRs, resulting in an almost entirely different set of contacts with MHC. Based on the structures of unbound G4 and E8, the distinct conformations of CDR1? in these TCRs are not induced by binding to mutTPI-DR1 but result from differences in the length and sequence of CDR3? that are transmitted to CDR1?. The editing of germ-line-encoded TCR-MHC interactions by CDR3 demonstrates that these interactions possess sufficient intrinsic flexibility to accommodate large structural variations in CDR3 and, consequently, in the TCR-binding site.
Project description:Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-? chain with the MHC class II ? chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3? loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3? loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.
Project description:We report crystal structures of a negatively selected T cell receptor (TCR) that recognizes two I-A(u)-restricted myelin basic protein peptides and one of its peptide/major histocompatibility complex (pMHC) ligands. Unusual complementarity-determining region (CDR) structural features revealed by our analyses identify a previously unrecognized mechanism by which the highly variable CDR3 regions define ligand specificity. In addition to the pMHC contact residues contributed by CDR3, the CDR3 residues buried deep within the V alpha/V beta interface exert indirect effects on recognition by influencing the V alpha/V beta interdomain angle. This phenomenon represents an additional mechanism for increasing the potential diversity of the TCR repertoire. Both the direct and indirect effects exerted by CDR residues can impact global TCR/MHC docking. Analysis of the available TCR structures in light of these results highlights the significance of the V alpha/V beta interdomain angle in determining specificity and indicates that TCR/pMHC interface features do not distinguish autoimmune from non-autoimmune class II-restricted TCRs.
Project description:The interaction between T-cell receptors (TCRs) of T-cells and potentially immunogenic peptides presented by MHCs of antigen presenting cells is one of the most important mechanisms of the adaptive human immune system. A large number of structural simulations of the TCR/peptide/MHC system have been carried out. However, to date no study has investigated the differences of the dynamics between free TCRs and pMHC bound TCRs on a large scale. Here we present a study totalling 37 100?ns investigating the LC13 TCR in its free form as well as in complex with HLA-B*08:01 and different peptides. Our results show that the dynamics of the bound and unbound LC13 TCR differ significantly. This is reflected in (a) expected results such as an increased flexibility and increased solvent accessible surface of the CDRs of unbound TCR simulations but also in (b) less expected results such as lower CDR distances and compactness as well as alteration in the hydrogen bond network around CDR3? of unbound TCR simulations. Our study further emphasises the structural flexibility of TCRs and confirms the importance of the CDR3 loops for the adoption to MHC.
Project description:alphabeta T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR-pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR-pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR-pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.
Project description:We have hypothesized that in the prenegative selection TCR repertoire, many somatically generated complementary-determining region (CDR) 3 loops combine with evolutionarily selected germline Valpha/Vbeta CDR1/CDR2 loops to create highly MHC/peptide cross-reactive T cells that are subsequently deleted by negative selection. Here, we present a mutational analysis of the Vbeta CDR3 of such a cross-reactive T-cell receptor (TCR), YAe62. Most YAe62 TCRs with the mutant CDR3s became less MHC promiscuous. However, others with CDR3s unrelated in sequence to the original recognized even more MHC alleles than the original TCR. Most importantly, this recognition was still dependent on the conserved CDR1/CDR2 residues. These results bolster the idea that germline TCR V elements are inherently reactive to MHC but that this reactivity is fine-tuned by the somatically generated CDR3 loops.
Project description:CD4 T lymphocyte activation requires T cell receptor (TCR) engagement by peptide/MHC (major histocompatibility complex) (pMHC). The TCR complementarity-determining region 3 (CDR3) contains variable ? and ? loops critical for pMHC recognition. During any immune response, tuning of TCR usage through progressive clonal selection occurs. Th1 and Th2 cells operate at different avidities for activation and display distinct transcriptional programs, although polarization may be plastic, influenced by pathogens and cytokines. We therefore hypothesized that CDR3?? sequence features may intrinsically influence CD4 phenotype during progression of a response.We show that CD4 polarization involves distinct CDR3? usage: Th1 and Th17 cells favored short TCR CDR3? sequences of 12 and 11 amino acids, respectively, while Th2 cells favored elongated CDR3? loops of 14 amino acids, with lower predicted affinity. The dominant Th2- and Th1-derived TCR? sequences with 14 amino acid CDR3 loops and 12 amino acid CDR3 loops, respectively, were expressed in TCR transgenics. The functional impact of these TCR? transgenes was assessed after in vivo priming with a peptide/adjuvant. The short, Th1-derived receptor transgenic T cell lines made IFN?, but not IL-4, 5 or 13, while the elongated, Th2-derived receptor transgenic T cell lines made little or no IFN?, but increased IL-4, 5 and 13 with progressive re-stimulations, mirrored by GATA-3 up-regulation. T cells from primed Th2 TCR? transgenics selected dominant TCR V? expansions, allowing us to generate TCR?? transgenics carrying the favored, Th2-derived receptor heterodimer. Primed T cells from TCR?? transgenics made little or no IL-17 or IFN?, but favored IL-9 after priming with Complete Freund's adjuvant and IL-4, 5, 9, 10 and 13 after priming with incomplete Freund's. In tetramer-binding studies, this transgenic receptor showed low binding avidity for pMHC and polarized T cell lines show TCR avidity for Th17 > Th1 > Th2. While transgenic expression of a Th2-derived, 'elongated' TCR-CDR3? and the TCR?? pair, clearly generated a program shifted away from Th1 immunity and with low binding avidity, cytokine-skewing could be over-ridden by altering peptide challenge dose.We propose that selection from responding clones with distinctive TCRs on the basis of functional avidity can direct a preference away from Th1 effector responses, favoring Th2 cytokines.