PMC 20201223 pmc.key 4887163 CC BY no 0 0 10.1172/JCI85679 4887163 27183389 85679 2191 6 This paper is licensed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2204 surname:Cole;given-names:David K. surname:Bulek;given-names:Anna M. surname:Rossjohn;given-names:Jamie surname:Zhu;given-names:Cheng surname:Miles;given-names:John J. surname:Peakman;given-names:Mark surname:Wooldridge;given-names:Linda surname:Rizkallah;given-names:Pierre J. surname:Sewell;given-names:Andrew K. surname:Dolton;given-names:Garry surname:Schauenberg;given-names:Andrea J. surname:Szomolay;given-names:Barbara surname:Rittase;given-names:William surname:Trimby;given-names:Andrew surname:Jothikumar;given-names:Prithiviraj surname:Fuller;given-names:Anna surname:Skowera;given-names:Ania TITLE front 126 2016 0 Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity 0.9898953 protein_type cleaner0 2023-07-19T20:52:37Z MESH: T cell receptor 0.9988882 chemical cleaner0 2023-07-20T07:57:25Z CHEBI: insulin ABSTRACT abstract 99 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. 0.9838819 complex_assembly cleaner0 2023-07-19T20:54:37Z GO: T cell antigen receptor 0.9382497 complex_assembly cleaner0 2023-07-19T20:53:35Z GO: TCR 0.9977353 complex_assembly cleaner0 2023-07-20T07:56:15Z GO: peptide–major histocompatibility complexes 0.99710053 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC 0.9991755 species cleaner0 2023-07-19T16:09:40Z MESH: human protein PR: cleaner0 2023-07-19T20:09:34Z preproinsulin complex_assembly GO: cleaner0 2023-07-20T07:49:27Z MHC 0.99922645 evidence cleaner0 2023-07-19T20:44:44Z DUMMY: structures complex_assembly GO: cleaner0 2023-07-19T20:54:50Z 1E6 TCR 0.999249 protein_state cleaner0 2023-07-19T16:13:16Z DUMMY: bound to chemical CHEBI: cleaner0 2023-07-19T20:10:47Z altered peptide ligands 0.9994267 evidence cleaner0 2023-07-19T20:44:48Z DUMMY: structures complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.80075455 evidence cleaner0 2023-07-19T20:44:51Z DUMMY: antipathogen-like binding affinity INTRO title_1 1445 Introduction INTRO paragraph 1458 T cells perform an essential role in adaptive immunity by interrogating the host proteome for anomalies, classically by recognizing peptides bound in major histocompatibility (MHC) molecules at the cell surface. Recent data supports the notion that, to perform this role, the highly variable αβ T cell antigen receptor (TCR) must be able to recognize thousands, if not millions, of different peptide ligands. This ability is required to enable the estimated 25 million distinct TCRs expressed in humans to provide effective immune coverage against all possible foreign peptide antigens. Although essential to avoid blind spots during pathogen recognition, T cell cross-reactivity has also been implicated as a pathway to autoimmunity, possibly mediated by highly reactive pathogen-specific T cells weakly recognizing self-ligands. 0.9515432 complex_assembly cleaner0 2023-07-20T07:49:14Z GO: major histocompatibility 0.9665209 complex_assembly cleaner0 2023-07-20T07:49:26Z GO: MHC 0.99938995 protein_state cleaner0 2023-07-20T07:59:48Z DUMMY: highly variable complex_assembly GO: cleaner0 2023-07-19T20:55:10Z αβ T cell antigen receptor 0.9842329 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.5031044 complex_assembly cleaner0 2023-07-19T20:55:23Z GO: TCRs 0.99924266 species cleaner0 2023-07-19T20:43:39Z MESH: humans INTRO paragraph 2296 Several mechanisms, by which TCRs could bind to a large number of different peptide-MHC (pMHC), have been proposed. Structures of unligated and ligated TCRs have shown that the TCR complementarity determining region (CDR) loops can be flexible, perhaps enabling peptide binding using different loop conformations. Both MHC and peptide have also been shown to undergo structural changes upon TCR binding, mediating an induced fit between the TCR and pMHC. Other studies, mainly in the murine system, have demonstrated that the same TCR can interact with different pMHCs using a common or divergent modality. Recent studies in model murine systems demonstrate that TCR cross-reactivity can be governed by recognition of a conserved region in the peptide that allows tolerance of peptide sequence variation outside of this hotspot. 0.9945287 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs complex_assembly GO: cleaner0 2023-07-19T16:24:59Z peptide-MHC 0.59893274 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC 0.9992618 evidence cleaner0 2023-07-19T20:44:54Z DUMMY: Structures 0.99964464 protein_state cleaner0 2023-07-19T16:17:41Z DUMMY: unligated 0.9996339 protein_state cleaner0 2023-07-19T20:05:05Z DUMMY: ligated 0.966741 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs 0.99959594 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.9739079 structure_element cleaner0 2023-07-19T16:28:16Z SO: complementarity determining region 0.999788 structure_element cleaner0 2023-07-19T16:28:27Z SO: CDR 0.99899834 structure_element cleaner0 2023-07-19T16:28:21Z SO: loops 0.9993062 structure_element cleaner0 2023-07-19T16:28:24Z SO: loop 0.40489408 complex_assembly cleaner0 2023-07-20T07:49:27Z GO: MHC 0.5937887 chemical cleaner0 2023-07-20T07:57:32Z CHEBI: peptide 0.80366987 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.9463298 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR complex_assembly GO: cleaner0 2023-07-19T16:24:26Z pMHC 0.99782526 taxonomy_domain cleaner0 2023-07-19T20:08:40Z DUMMY: murine 0.9772652 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.45551145 complex_assembly cleaner0 2023-07-19T16:28:10Z GO: pMHCs 0.99696213 taxonomy_domain cleaner0 2023-07-19T20:08:40Z DUMMY: murine 0.9929351 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR INTRO paragraph 3125 We recently reported that the 1E6 human CD8+ T cell clone — which mediates the destruction of β cells through the recognition of a major, HLA-A*0201–restricted, preproinsulin signal peptide (ALWGPDPAAA15–24) — can recognize upwards of 1 million different peptides. CD8+ T cells that recognize HLA-A*0201–ALWGPDPAAA have been shown to populate insulitic lesions in patients with type 1 diabetes (T1D). We demonstrated that the TCR from the 1E6 T cell clone bound to HLA-A*0201–ALWGPDPAAA using a limited footprint and very weak binding affinity. This first experimental evidence of a high level of CD8+ T cell cross-reactivity in a human autoimmune disease system hinted toward molecular mimicry by a more potent pathogenic peptide as a potential mechanism leading to β cell destruction. Here, we solved the structure of the 1E6 TCR with 7 altered peptide ligands (APLs) determined by our previously published combinatorial peptide library (CPL) screening, 2 of which mapped within human pathogens. These APLs differed from the natural preproinsulin peptide by up to 7 of 10 residues. We also solved the structure of each unligated APL to investigate whether structural changes occurred before or after binding — which, combined with an in-depth cellular and biophysical analysis of the 1E6 interaction with each APL, demonstrated the molecular mechanism mediating the high level of cross-reactivity exhibited by this preproinsulin-reactive human CD8+ T cell clone. 0.9989231 species cleaner0 2023-07-19T16:09:40Z MESH: human protein PR: cleaner0 2023-07-19T16:27:26Z HLA-A*0201 0.75353974 protein cleaner0 2023-07-19T20:09:34Z PR: preproinsulin 0.8835323 structure_element cleaner0 2023-07-20T08:09:34Z SO: signal peptide 0.5604127 chemical cleaner0 2023-07-19T20:09:47Z CHEBI: ALWGPDPAAA15–24 0.98721063 complex_assembly cleaner0 2023-07-19T16:27:47Z GO: HLA-A*0201–ALWGPDPAAA 0.662854 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.99923784 protein_state cleaner0 2023-07-19T16:13:16Z DUMMY: bound to 0.946986 complex_assembly cleaner0 2023-07-19T16:27:48Z GO: HLA-A*0201–ALWGPDPAAA 0.9991387 evidence cleaner0 2023-07-19T16:20:13Z DUMMY: binding affinity 0.99896324 species cleaner0 2023-07-19T16:09:40Z MESH: human 0.99941254 experimental_method cleaner0 2023-07-20T08:05:04Z MESH: solved 0.9995402 evidence cleaner0 2023-07-19T20:44:58Z DUMMY: structure complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR chemical CHEBI: cleaner0 2023-07-19T16:29:05Z altered peptide ligands 0.9808608 chemical cleaner0 2023-07-19T16:29:09Z CHEBI: APLs experimental_method MESH: cleaner0 2023-07-20T08:05:31Z combinatorial peptide library (CPL) screening 0.9985892 species cleaner0 2023-07-19T16:09:40Z MESH: human 0.8167679 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.9198488 protein cleaner0 2023-07-19T20:09:34Z PR: preproinsulin 0.9994512 experimental_method cleaner0 2023-07-20T08:05:40Z MESH: solved 0.999616 evidence cleaner0 2023-07-19T20:45:01Z DUMMY: structure 0.99964905 protein_state cleaner0 2023-07-19T16:17:41Z DUMMY: unligated 0.9893568 chemical cleaner0 2023-07-19T16:29:16Z CHEBI: APL 0.9985127 experimental_method cleaner0 2023-07-20T08:05:43Z MESH: cellular and biophysical analysis 0.97832096 chemical cleaner0 2023-07-19T16:29:15Z CHEBI: APL protein PR: cleaner0 2023-07-19T20:09:34Z preproinsulin 0.9987363 species cleaner0 2023-07-19T16:09:40Z MESH: human RESULTS title_1 4606 Results RESULTS title_2 4614 The 1E6 T cell clone recognizes APLs across a large dynamic range. 0.9728898 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs RESULTS paragraph 4681 We have previously demonstrated that the 1E6 T cell clone can recognize over 1 million different peptides with a potency comparable with, or better than, the cognate preproinsulin peptide ALWGPDPAAA. From this large functional scan, we selected 7 different APLs that activated the 1E6 T cell clone across a wide (4-log) functional range (Table 1). Two of these peptides, MVWGPDPLYV and RQFGPDWIVA (bold text signifies amino acids that are different from the index preproinsulin–derived sequence), are contained within the proteomes of the human pathogens Bacteroides fragilis/thetaiotaomicron and Clostridium asparagiforme, respectively. Competitive functional testing revealed that the preproinsulin-derived sequence ALWGPDPAAA was one of the least potent targets for 1E6, with only the MVWGPDPLYV and YLGGPDFPTI demonstrating a similar low-activity profile in MIP-1β secretion and target killing assays (Figure 1, A and B). The RQFGPDWIVA sequence (present in C. asparagiforme) activated the 1E6 T cell with around 1 log–greater potency compared with ALWGPDPAAA. At the other end of the spectrum, the RQFGPDFPTI peptide stimulated MIP-1β release and killing by 1E6 at an exogenous peptide concentration 2–3 logs lower compared with ALWGPDPAAA. The pattern of peptide potency was closely mirrored by pMHC tetramer staining experiments (Figure 1C and plots shown in Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI85679DS1). Here, the A2-RQFGPDFPTI tetramer stained 1E6 with the greatest MFI, gradually decreasing to the weakest tetramers: A2-MVWGPDPLYV and -YLGGPDFPTI. To parallel the functional analysis, we also performed thermal melt (Tm) experiments using synchrotron radiation circular dichroism (SRCD) to investigate the stability of each APL (Figure 1D). The range of Tm was between 49.4°C (RQFGPDWIVA) and 60.3°C (YQFGPDFPIA), with an average approximately 55°C, similar to our previous findings. This pattern of stability did not correlate with the T cell activation or tetramer staining experiments, indicating that peptide binding to the MHC do not explain ligand potency. 0.9016905 protein cleaner0 2023-07-19T20:09:35Z PR: preproinsulin 0.7047767 chemical cleaner0 2023-07-19T16:08:19Z CHEBI: ALWGPDPAAA 0.9961451 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.98260164 chemical cleaner0 2023-07-19T16:08:35Z CHEBI: MVWGPDPLYV 0.96969604 chemical cleaner0 2023-07-19T16:08:55Z CHEBI: RQFGPDWIVA 0.8399371 protein cleaner0 2023-07-19T20:09:35Z PR: preproinsulin 0.9994413 species cleaner0 2023-07-19T16:09:39Z MESH: human 0.9981843 species cleaner0 2023-07-19T16:09:27Z MESH: Bacteroides fragilis/thetaiotaomicron 0.9994335 species cleaner0 2023-07-19T16:09:22Z MESH: Clostridium asparagiforme 0.999571 experimental_method cleaner0 2023-07-20T08:06:01Z MESH: Competitive functional testing 0.55834925 protein cleaner0 2023-07-19T20:09:35Z PR: preproinsulin 0.8603277 chemical cleaner0 2023-07-19T16:08:19Z CHEBI: ALWGPDPAAA 0.9908454 chemical cleaner0 2023-07-19T16:08:36Z CHEBI: MVWGPDPLYV 0.89793074 chemical cleaner0 2023-07-19T16:09:09Z CHEBI: YLGGPDFPTI protein PR: cleaner0 2023-07-19T20:12:00Z MIP-1β 0.9913579 chemical cleaner0 2023-07-19T16:08:56Z CHEBI: RQFGPDWIVA 0.99943805 species cleaner0 2023-07-19T16:09:33Z MESH: C. asparagiforme 0.89824975 chemical cleaner0 2023-07-19T16:08:19Z CHEBI: ALWGPDPAAA 0.9736738 chemical cleaner0 2023-07-19T16:30:12Z CHEBI: RQFGPDFPTI protein PR: cleaner0 2023-07-19T20:12:01Z MIP-1β 0.811304 chemical cleaner0 2023-07-19T16:08:19Z CHEBI: ALWGPDPAAA 0.7686303 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC 0.97690034 experimental_method cleaner0 2023-07-20T08:06:08Z MESH: tetramer staining 0.834167 chemical cleaner0 2023-07-19T16:12:27Z CHEBI: A2-RQFGPDFPTI 0.7076429 oligomeric_state cleaner0 2023-07-20T07:58:17Z DUMMY: tetramer 0.9878656 oligomeric_state cleaner0 2023-07-20T07:58:19Z DUMMY: tetramers 0.9034901 chemical cleaner0 2023-07-19T16:12:10Z CHEBI: A2-MVWGPDPLYV 0.5810209 chemical cleaner0 2023-07-19T16:09:10Z CHEBI: YLGGPDFPTI 0.999585 experimental_method cleaner0 2023-07-20T08:06:17Z MESH: thermal melt 0.9986412 evidence cleaner0 2023-07-19T16:29:57Z DUMMY: Tm 0.99956584 experimental_method cleaner0 2023-07-20T08:06:25Z MESH: synchrotron radiation circular dichroism 0.9995995 experimental_method cleaner0 2023-07-20T08:06:28Z MESH: SRCD 0.99415684 chemical cleaner0 2023-07-19T16:29:16Z CHEBI: APL 0.99952316 evidence cleaner0 2023-07-19T16:29:58Z DUMMY: Tm 0.7603012 chemical cleaner0 2023-07-19T16:08:56Z CHEBI: RQFGPDWIVA 0.9138942 chemical cleaner0 2023-07-19T20:12:21Z CHEBI: YQFGPDFPIA 0.9968275 experimental_method cleaner0 2023-07-20T08:06:31Z MESH: tetramer staining 0.9852401 complex_assembly cleaner0 2023-07-20T07:49:27Z GO: MHC RESULTS title_2 6826 The 1E6 TCR can bind peptides with strong antipathogen-like affinities. complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR RESULTS paragraph 6898 We, and others, have previously demonstrated that antipathogenic TCRs tend to bind with stronger affinity compared with self-reactive TCRs, likely a consequence of the deletion of T cells with high-affinity self-reactive TCR during thymic selection. In accordance with this trend, the 1E6 TCR bound the natural preproinsulin peptide, ALWGPDPAAA, with the weakest affinity currently published for a human CD8+ T cell–derived TCR with a biologically relevant ligand (KD > 200 μM; KD, equilibrium binding constant). Surface plasmon resonance (SPR) analysis of the 1E6 TCR–pMHC interaction for all 7 APLs (Figure 2, A–H) demonstrated that stronger binding affinity (represented as ΔG°, kcal/mol) correlated well with the EC50 values (peptide concentration required to reach half-maximal 1E6 T cell killing) for each ligand, demonstrated by a Pearson’s correlation analysis value of 0.8 (P = 0.01) (Figure 2I). It should be noted that this correlation, although consistent with the T cell killing experiments, uses only approximate affinities calculated for the 2 weakest ligands. These experiments revealed 4 important findings. First, the 1E6 T cell could still functionally respond to peptide when the TCR binding affinity was extremely weak, e.g., the 1E6 TCR binding affinity for the A2-MVWGPDPLYV peptide was KD = ~600 μM. Second, the 1E6 TCR bound to A2-RQFGPDFPTI with KD = 0.5 μM, equivalent to the binding affinity of the very strongest antipathogen TCRs. Third, the 1E6 TCR bound to A2-RQFGPDWIVA peptide, within the C. asparagiforme proteome, with approximately 4-fold stronger affinity than A2-ALWGPDPAAA, demonstrating the potential for a pathogen-derived antigen to initiate a response to the self-derived sequence. Finally, these data demonstrate the largest range of binding affinities reported for a natural, endogenous human TCR of more than 3 logs of magnitude (A2-MVWGPDPLYV vs. A2-RQFGPDFPTI). To confirm the affinity spread detected by SPR, and to evaluate whether experiments performed using soluble molecules were biologically relevant to events at the T cell surface, we determined the effective 2D affinity of each APL using an adhesion frequency assay in which a human rbc coated in pMHC acted as an adhesion sensor. In agreement with SPR experiments, the range of 2D affinities we detected differed by around 3 logs, with the A2-MVWGPDPLYV generating the weakest 2D affinity (2.6 × 10–5 AcKa μm4) and A2-RQFGPDFPTI the strongest (4.5 × 10–2 AcKa μm4) (Figure 2J). As with the 3D affinity measurements, the 2D affinity measurements correlated well with the EC50 values for each ligand (Figure 2K) demonstrating a strong correlation (Pearson’s correlation = 0.8, P = 0.01) between T cell antigen sensitivity and TCR binding affinity. Of note, these data demonstrate a close agreement between the 3D affinity values generated using SPR and 2D affinity values generated using adhesion frequency assays. 0.9443769 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.9800795 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.9109045 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9852579 protein_state cleaner0 2023-07-19T20:56:07Z DUMMY: bound 0.54183936 protein cleaner0 2023-07-19T20:09:35Z PR: preproinsulin 0.9975931 chemical cleaner0 2023-07-19T16:08:19Z CHEBI: ALWGPDPAAA 0.87047696 evidence cleaner0 2023-07-19T16:19:49Z DUMMY: affinity 0.99915814 species cleaner0 2023-07-19T16:09:40Z MESH: human 0.99290437 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.9996025 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.99964154 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.9994817 evidence cleaner0 2023-07-19T18:02:09Z DUMMY: equilibrium binding constant 0.99956703 experimental_method cleaner0 2023-07-19T18:02:05Z MESH: Surface plasmon resonance 0.9996811 experimental_method cleaner0 2023-07-19T16:32:30Z MESH: SPR complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR complex_assembly GO: cleaner0 2023-07-19T16:24:26Z pMHC 0.9805093 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.999523 evidence cleaner0 2023-07-19T16:20:13Z DUMMY: binding affinity 0.99963284 evidence cleaner0 2023-07-19T20:15:38Z DUMMY: ΔG° 0.9974759 evidence cleaner0 2023-07-19T20:45:05Z DUMMY: EC50 0.99587667 experimental_method cleaner0 2023-07-19T16:31:19Z MESH: Pearson’s correlation analysis 0.9993838 evidence cleaner0 2023-07-19T20:45:09Z DUMMY: affinities evidence DUMMY: cleaner0 2023-07-19T18:06:46Z TCR binding affinity evidence DUMMY: cleaner0 2023-07-19T18:08:11Z 1E6 TCR binding affinity 0.99747103 chemical cleaner0 2023-07-19T16:12:02Z CHEBI: A2-MVWGPDPLYV 0.99966264 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9989283 protein_state cleaner0 2023-07-19T16:13:16Z DUMMY: bound to 0.9977234 chemical cleaner0 2023-07-19T16:12:27Z CHEBI: A2-RQFGPDFPTI 0.99965525 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.99953234 evidence cleaner0 2023-07-19T16:20:13Z DUMMY: binding affinity 0.98072755 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9973413 protein_state cleaner0 2023-07-19T16:13:16Z DUMMY: bound to 0.9959906 chemical cleaner0 2023-07-19T16:12:47Z CHEBI: A2-RQFGPDWIVA 0.99946743 species cleaner0 2023-07-19T16:09:34Z MESH: C. asparagiforme 0.9960381 evidence cleaner0 2023-07-19T16:19:49Z DUMMY: affinity 0.9956104 chemical cleaner0 2023-07-19T16:16:49Z CHEBI: A2-ALWGPDPAAA 0.9995508 evidence cleaner0 2023-07-19T20:45:17Z DUMMY: binding affinities 0.70571 protein_state cleaner0 2023-07-20T08:00:03Z DUMMY: endogenous 0.9993166 species cleaner0 2023-07-19T16:09:40Z MESH: human 0.9945815 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.9969234 chemical cleaner0 2023-07-19T16:12:10Z CHEBI: A2-MVWGPDPLYV 0.9975858 chemical cleaner0 2023-07-19T16:12:27Z CHEBI: A2-RQFGPDFPTI evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.99969566 experimental_method cleaner0 2023-07-19T16:32:29Z MESH: SPR 0.9962995 evidence cleaner0 2023-07-19T16:32:21Z DUMMY: 2D affinity 0.9885676 chemical cleaner0 2023-07-19T16:29:16Z CHEBI: APL 0.999513 experimental_method cleaner0 2023-07-19T20:12:51Z MESH: adhesion frequency assay 0.99917644 species cleaner0 2023-07-19T16:09:40Z MESH: human 0.76752275 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC 0.99969506 experimental_method cleaner0 2023-07-19T16:32:30Z MESH: SPR evidence DUMMY: cleaner0 2023-07-19T16:32:46Z 2D affinities chemical CHEBI: cleaner0 2023-07-19T16:12:10Z A2-MVWGPDPLYV evidence DUMMY: cleaner0 2023-07-19T16:32:22Z 2D affinity evidence DUMMY: cleaner0 2023-07-19T20:51:27Z AcKa chemical CHEBI: cleaner0 2023-07-19T16:12:27Z A2-RQFGPDFPTI evidence DUMMY: cleaner0 2023-07-19T20:51:36Z AcKa evidence DUMMY: cleaner0 2023-07-19T16:33:07Z 3D affinity evidence DUMMY: cleaner0 2023-07-19T16:32:22Z 2D affinity evidence DUMMY: cleaner0 2023-07-19T20:51:31Z EC50 evidence DUMMY: cleaner0 2023-07-19T16:14:28Z Pearson’s correlation evidence DUMMY: cleaner0 2023-07-19T20:51:21Z P evidence DUMMY: cleaner0 2023-07-19T18:06:46Z TCR binding affinity evidence DUMMY: cleaner0 2023-07-19T16:33:03Z 3D affinity experimental_method MESH: cleaner0 2023-07-19T16:32:30Z SPR evidence DUMMY: cleaner0 2023-07-19T16:32:22Z 2D affinity RESULTS title_2 9844 The 1E6 TCR uses a consensus binding mode to engage multiple APLs. complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.51438355 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs RESULTS paragraph 9911 Our previous structure of the 1E6-A2-ALWGPDPAAA complex demonstrated a limited binding footprint between the TCR and pMHC. The low number of contacts between the 2 molecules most likely contributed to the weak binding affinity of the interaction. In order to examine the mechanism by which the 1E6 TCR engaged a wide range of peptides with divergent binding affinities, we solved the structure of the 1E6 TCR in complex with all 7 APLs used in Figure 2. All structures were solved in space group P1 to 2–3 Å resolution with crystallographic Rwork/Rfree ratios within accepted limits as shown in the theoretically expected distribution (ref. and Supplemental Table 1). The 1E6 TCR used a very similar overall binding modality to engage all of the APLs, with root mean square deviation ranging between 0.81 and 1.12 Å2 (compared with 1E6-A2-ALWGPDPAAA). The relatively broad range of buried surface areas (1,670–1,920 Å2) did not correlate well with TCR binding affinity (Pearson’s correlation = 0.45, P = 0.2). The surface complementarity values (0.52–0.7) correlated slightly with affinity (Pearson’s correlation = 0.7, P = 0.05) but could not explain all differences in binding (Figure 3A and Table 2). The TCR CDR loops were in a very similar position in all complexes, apart from some slight deviations in the TCR β-chain (Figure 3B); the peptides were all presented in a similar conformation (Figure 3C); and there was minimal variation in crossing angles of the TCR (42.3°–45.6°) (Figure 3D). Overall, the 1E6 TCR used a canonical binding mode to engage each APL with the TCR α-chain positioned over the MHC class I (MHCI) α2-helix and the TCR β-chain over the MHCI α-1 helix, straddling the peptide cargo. However, subtle differences in the respective interfaces were apparent (discussed below) and resulted in altered binding affinities of the respective complexes. 0.9994141 evidence cleaner0 2023-07-19T20:13:22Z DUMMY: structure 0.9996247 complex_assembly cleaner0 2023-07-19T16:14:42Z GO: 1E6-A2-ALWGPDPAAA 0.8440795 site cleaner0 2023-07-20T08:12:19Z SO: binding footprint 0.66571075 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.86722314 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC 0.9874364 evidence cleaner0 2023-07-19T16:20:13Z DUMMY: binding affinity complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.5872233 evidence cleaner0 2023-07-19T20:45:24Z DUMMY: binding affinities 0.99880445 experimental_method cleaner0 2023-07-20T08:06:37Z MESH: solved 0.99947244 evidence cleaner0 2023-07-19T20:13:19Z DUMMY: structure complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.99926734 protein_state cleaner0 2023-07-19T18:02:49Z DUMMY: in complex with 0.74618053 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.9814917 evidence cleaner0 2023-07-19T20:13:14Z DUMMY: structures 0.7286678 experimental_method cleaner0 2023-07-20T08:06:42Z MESH: solved 0.99763364 evidence cleaner0 2023-07-19T20:13:11Z DUMMY: Rwork/Rfree ratios complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.5221809 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.9993834 evidence cleaner0 2023-07-19T20:13:17Z DUMMY: root mean square deviation 0.99953204 complex_assembly cleaner0 2023-07-19T16:14:43Z GO: 1E6-A2-ALWGPDPAAA 0.87155503 evidence cleaner0 2023-07-19T17:58:44Z DUMMY: TCR binding affinity evidence DUMMY: cleaner0 2023-07-19T16:14:23Z Pearson’s correlation evidence DUMMY: cleaner0 2023-07-19T20:51:59Z P 0.99492294 evidence cleaner0 2023-07-19T20:13:26Z DUMMY: surface complementarity values evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity evidence DUMMY: cleaner0 2023-07-19T16:14:28Z Pearson’s correlation evidence DUMMY: cleaner0 2023-07-19T20:52:03Z P 0.5172308 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.9997113 structure_element cleaner0 2023-07-19T17:58:59Z SO: CDR loops 0.5146105 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.99949646 structure_element cleaner0 2023-07-19T17:59:47Z SO: β-chain 0.92718446 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.5723033 chemical cleaner0 2023-07-19T16:29:16Z CHEBI: APL 0.72113127 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.99952 structure_element cleaner0 2023-07-19T17:59:52Z SO: α-chain complex_assembly GO: cleaner0 2023-07-20T07:49:55Z MHC class I 0.9365372 complex_assembly cleaner0 2023-07-20T07:50:06Z GO: MHCI 0.99968386 structure_element cleaner0 2023-07-20T08:09:40Z SO: α2-helix 0.5055066 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.99942774 structure_element cleaner0 2023-07-19T17:59:48Z SO: β-chain 0.5436469 complex_assembly cleaner0 2023-07-20T07:50:07Z GO: MHCI 0.9996726 structure_element cleaner0 2023-07-20T08:09:44Z SO: α-1 helix 0.99937385 site cleaner0 2023-07-20T08:12:23Z SO: interfaces 0.9975358 evidence cleaner0 2023-07-19T20:45:30Z DUMMY: binding affinities RESULTS title_2 11806 Interactions between the 1E6 TCR and different APLs are focused around a conserved GPD peptide motif. complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9785179 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.99952507 protein_state cleaner0 2023-07-20T08:00:12Z DUMMY: conserved structure_element SO: cleaner0 2023-07-19T18:04:37Z GPD peptide motif RESULTS paragraph 11908 We next performed an in-depth atomic analysis of the contacts between the 1E6 TCR and each APL to determine the structural basis for the altered T cell peptide sensitivities and TCR binding affinities (Table 2). Concomitant with our global analysis of 1E6 TCR binding to the APLs, we observed a common interaction element, consistent with our previous findings, that utilized TCR residues Tyr97α and Trp97β, forming an aromatic cap over a central GPD motif that was present in all of the APLs (Figure 4). Interactions between these 2 TCR and 3 peptide residues accounted for 41%–50% of the total contacts across all complexes (Table 2), demonstrating the conserved peptide centric binding mode utilized by the 1E6 TCR. This fixed anchoring between the 2 molecules was important for stabilization of the TCR-pMHC complex, as — although other peptides without the ‘GDP’ motif were tested and shown to activate the 1E6 T cell clone — we were unable to measure robust affinities using SPR (data not shown). These data support the requirement for a conserved interaction between the 1E6 TCR and the GPD motif, as we observed in our previously published 1E6-A2-ALWGPDPAAA structure. 0.94664764 experimental_method cleaner0 2023-07-20T08:06:47Z MESH: atomic analysis complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.7138178 chemical cleaner0 2023-07-19T16:29:16Z CHEBI: APL 0.9993618 evidence cleaner0 2023-07-19T18:03:25Z DUMMY: TCR binding affinities complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.7065273 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.9956772 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.99990237 residue_name_number cleaner0 2023-07-19T18:00:25Z DUMMY: Tyr97α 0.9999002 residue_name_number cleaner0 2023-07-19T18:00:43Z DUMMY: Trp97β 0.8847289 structure_element cleaner0 2023-07-20T08:09:51Z SO: aromatic cap 0.9996864 structure_element cleaner0 2023-07-19T18:05:06Z SO: GPD motif 0.49906126 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.99199945 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.9910685 protein_state cleaner0 2023-07-19T20:52:22Z DUMMY: conserved complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.99948066 complex_assembly cleaner0 2023-07-19T18:04:07Z GO: TCR-pMHC structure_element SO: cleaner0 2023-07-19T18:05:00Z ‘GDP’ motif 0.99936527 evidence cleaner0 2023-07-19T20:13:53Z DUMMY: affinities 0.99967253 experimental_method cleaner0 2023-07-19T16:32:30Z MESH: SPR 0.9992118 protein_state cleaner0 2023-07-19T20:52:25Z DUMMY: conserved complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.99967885 structure_element cleaner0 2023-07-19T18:05:06Z SO: GPD motif 0.9997021 complex_assembly cleaner0 2023-07-19T16:14:43Z GO: 1E6-A2-ALWGPDPAAA 0.9996151 evidence cleaner0 2023-07-19T20:13:48Z DUMMY: structure RESULTS title_2 13097 Focused hotspot binding around a conserved GPD motif enables the 1E6 TCR to tolerate peptide degeneracy. 0.99951005 protein_state cleaner0 2023-07-20T08:00:19Z DUMMY: conserved 0.9997299 structure_element cleaner0 2023-07-19T18:05:05Z SO: GPD motif complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR RESULTS paragraph 13202 Although the 1E6 TCR formed a similar overall interaction with each APL, the stabilization between the TCR and the GPD motif enabled fine differences in the contact network with both the peptide and MHC surface that allowed discrimination between each ligand (Figure 5). For example, the 1E6 TCR made only 47 peptide contacts with A2-MVWGPDPLYV (KD = ~600 μM) compared with 63 and 57 contacts with A2-YQFGPDFPIA (KD = 7.4 μM) and A2-RQFGPDFPTI (KD = 0.5 μM), respectively. Although the number of peptide contacts was a good predictor of TCR binding affinity for some of the APLs, for others, the correlation was poor (Pearson’s correlation = 0.045, P = 0.92), possibly because of different resolutions for each complex structure. For example, the 1E6 TCR made 64 peptide contacts with A2-YLGGPDFPTI (KD = ~400 μM) compared with 43 contacts with A2-RQWGPDPAAV (KD = 7.8 μM). complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9926323 chemical cleaner0 2023-07-19T16:29:16Z CHEBI: APL 0.9984389 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.99968 structure_element cleaner0 2023-07-19T18:05:06Z SO: GPD motif 0.99865997 site cleaner0 2023-07-20T08:12:28Z SO: contact network 0.9031253 chemical cleaner0 2023-07-19T20:14:20Z CHEBI: peptide 0.9292942 site cleaner0 2023-07-20T07:50:45Z SO: MHC surface complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9980581 chemical cleaner0 2023-07-19T16:12:10Z CHEBI: A2-MVWGPDPLYV 0.9408846 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.998652 chemical cleaner0 2023-07-19T16:15:51Z CHEBI: A2-YQFGPDFPIA 0.97073865 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.9988263 chemical cleaner0 2023-07-19T16:12:27Z CHEBI: A2-RQFGPDFPTI 0.83079475 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.99772674 evidence cleaner0 2023-07-19T18:06:16Z DUMMY: TCR binding affinity 0.99602747 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.9775787 evidence cleaner0 2023-07-19T16:14:28Z DUMMY: Pearson’s correlation 0.9996146 evidence cleaner0 2023-07-19T20:14:33Z DUMMY: structure complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9981677 chemical cleaner0 2023-07-19T16:16:10Z CHEBI: A2-YLGGPDFPTI 0.9152873 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.9987187 chemical cleaner0 2023-07-19T16:16:23Z CHEBI: A2-RQWGPDPAAV 0.9516478 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD RESULTS paragraph 14083 The most important peptide modification in terms of generating new contacts was peptide position 1. The stronger ligands all encoded larger side chains (Arg or Tyr) at peptide position 1 (Figure 5, E–H), enabling interactions with 1E6 that were not present in the weaker APLs that lacked large side chains in this position (Figure 5, A, C, and D). We have previously shown that the 1E6 TCR uses a rigid lock-and-key mechanism during binding to A2-ALWGPDPAAA. These data demonstrated that the unligated structure of the 1E6 TCR was virtually identical to its ligated counterparts. In order to determine whether any of the APLs required an induced fit mechanism during binding that could explain the difference in free binding energy (ΔG) between each complex (Table 2), we solved the unligated structures of all 7 APLs (the A2-ALWGPDPAAA structure has been previously published and was used in this comparison, ref.) (Figure 6 and Supplemental Table 2). The unligated A2-MVWGPDPLYV (KD = ~600 μM) structure revealed that the side chain Tyr9 swung around 8 Å in the complex structure, subsequently making contacts with TCR residues Asp30β and Asn51β (Figure 6A and Figure 5A, respectively). This movement could result in an entropic penalty contributing to the weak TCR binding affinity we observed for this ligand. Additional small movements in the Cα backbone of the peptide around peptide residue Asp6 were apparent in the A2-YLGGPDFPTI (KD = ~400 μM), A2-ALWGPDPAAA (KD = ~208 μM), and A2-RQFGPDWIVA (KD = 44.4 μM) structures (Figure 6, B, C, and E). The unligated structures of A2-AQWGPDAAA, A2-RQWGPDPAAV, A2-YQFGPDFPIA, and A2-RQFGPDFPTI were virtually identical when in complex with 1E6 (Figure 6, D and F–H). Apart from the case of A2-AQWGPDAAA (KD = 61.9 μM), these observations support the conclusion that the higher-affinity ligands required less conformational melding during binding, which could be energetically beneficial (lower entopic cost) during ligation with the 1E6 TCR. 0.98920166 residue_number cleaner0 2023-07-20T08:04:04Z DUMMY: 1 0.99930096 residue_name cleaner0 2023-07-19T20:03:56Z SO: Arg 0.99929297 residue_name cleaner0 2023-07-19T20:03:59Z SO: Tyr 0.98596257 residue_number cleaner0 2023-07-20T08:04:08Z DUMMY: 1 0.529526 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9851528 chemical cleaner0 2023-07-19T16:16:49Z CHEBI: A2-ALWGPDPAAA 0.9996426 protein_state cleaner0 2023-07-19T16:17:41Z DUMMY: unligated 0.99905044 evidence cleaner0 2023-07-19T20:14:36Z DUMMY: structure complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.99959403 protein_state cleaner0 2023-07-19T20:05:05Z DUMMY: ligated chemical CHEBI: cleaner0 2023-07-19T16:29:10Z APLs 0.99935204 evidence cleaner0 2023-07-19T20:14:40Z DUMMY: free binding energy 0.9987571 evidence cleaner0 2023-07-19T20:14:44Z DUMMY: ΔG 0.99803597 experimental_method cleaner0 2023-07-20T07:50:57Z MESH: solved 0.99954385 protein_state cleaner0 2023-07-19T16:17:41Z DUMMY: unligated 0.99930704 evidence cleaner0 2023-07-19T20:14:38Z DUMMY: structures 0.6730447 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.9470319 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.99931955 evidence cleaner0 2023-07-19T20:45:35Z DUMMY: structure 0.99965835 protein_state cleaner0 2023-07-19T16:17:40Z DUMMY: unligated 0.9856176 chemical cleaner0 2023-07-19T16:12:10Z CHEBI: A2-MVWGPDPLYV 0.99568236 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.99956495 evidence cleaner0 2023-07-19T20:14:51Z DUMMY: structure 0.9999052 residue_name_number cleaner0 2023-07-19T20:04:10Z DUMMY: Tyr9 0.9995851 evidence cleaner0 2023-07-19T20:14:54Z DUMMY: structure 0.87050486 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.9999012 residue_name_number cleaner0 2023-07-19T20:04:14Z DUMMY: Asp30β 0.9999014 residue_name_number cleaner0 2023-07-19T20:04:16Z DUMMY: Asn51β 0.99930316 evidence cleaner0 2023-07-19T18:06:45Z DUMMY: TCR binding affinity 0.99990344 residue_name_number cleaner0 2023-07-19T20:04:19Z DUMMY: Asp6 0.98224705 chemical cleaner0 2023-07-19T16:16:10Z CHEBI: A2-YLGGPDFPTI 0.98664665 evidence cleaner0 2023-07-19T16:17:16Z DUMMY: KD 0.97232896 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.9939036 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.9855407 chemical cleaner0 2023-07-19T16:12:47Z CHEBI: A2-RQFGPDWIVA 0.9931004 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.99954164 evidence cleaner0 2023-07-19T20:14:57Z DUMMY: structures 0.999648 protein_state cleaner0 2023-07-19T16:17:41Z DUMMY: unligated 0.99961734 evidence cleaner0 2023-07-19T20:15:00Z DUMMY: structures 0.98120284 chemical cleaner0 2023-07-19T16:17:31Z CHEBI: A2-AQWGPDAAA 0.96967775 chemical cleaner0 2023-07-19T16:16:24Z CHEBI: A2-RQWGPDPAAV 0.9569805 chemical cleaner0 2023-07-19T16:15:52Z CHEBI: A2-YQFGPDFPIA 0.9750207 chemical cleaner0 2023-07-19T16:12:27Z CHEBI: A2-RQFGPDFPTI 0.9942916 protein_state cleaner0 2023-07-19T18:02:49Z DUMMY: in complex with 0.9857505 chemical cleaner0 2023-07-19T16:17:32Z CHEBI: A2-AQWGPDAAA 0.9792589 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR RESULTS title_2 16093 Peptide modifications alter the interaction between the 1E6 TCR and the MHC surface. complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.98852634 site cleaner0 2023-07-20T07:51:05Z SO: MHC surface RESULTS paragraph 16178 In addition to changes between the TCR and peptide component, we also observed that different APLs had different knock-on effects between the TCR and MHC. MHC residue Arg65 that forms part of the MHC restriction triad (Arg65, Ala69, and Gln155) played a central role in TCR-MHC contacts, with Gln155 playing a less important role and Ala69 playing no role in binding at the interface (Figure 7). Generally, the weaker-affinity APLs made fewer contacts with the MHC surface (27–29 interactions) compared with the stronger-affinity APLs (29–35 contacts), consistent with a better Pearson’s correlation value (0.55) compared with TCR-peptide interactions versus affinity (0.045). For instance, contacts were made between TCR residue Val53β and MHC residue Gln72 in all APLs except for in the weakest affinity ligand pair, 1E6-A2-MVWGPDPLYV, in which a subtle change in TCR conformation — probably mediated by different peptide contacts — abrogated this interaction (Figure 7A). 0.9057284 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR chemical CHEBI: cleaner0 2023-07-19T16:29:10Z APLs 0.98210955 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.9670497 complex_assembly cleaner0 2023-07-20T07:49:27Z GO: MHC 0.9803191 complex_assembly cleaner0 2023-07-20T07:49:27Z GO: MHC 0.99990535 residue_name_number cleaner0 2023-07-19T18:07:15Z DUMMY: Arg65 0.9910882 site cleaner0 2023-07-20T07:51:19Z SO: MHC restriction triad 0.9999044 residue_name_number cleaner0 2023-07-19T18:07:15Z DUMMY: Arg65 0.9999025 residue_name_number cleaner0 2023-07-19T18:07:21Z DUMMY: Ala69 0.9999012 residue_name_number cleaner0 2023-07-19T18:07:25Z DUMMY: Gln155 0.6897632 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR complex_assembly GO: cleaner0 2023-07-20T07:49:27Z MHC 0.9999025 residue_name_number cleaner0 2023-07-19T18:07:26Z DUMMY: Gln155 0.9999045 residue_name_number cleaner0 2023-07-19T18:07:21Z DUMMY: Ala69 0.99446267 site cleaner0 2023-07-20T08:12:34Z SO: interface evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.9721638 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.98300236 complex_assembly cleaner0 2023-07-20T07:49:27Z GO: MHC evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.9680678 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.9995654 evidence cleaner0 2023-07-19T20:45:41Z DUMMY: Pearson’s correlation value 0.40218425 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.9993118 evidence cleaner0 2023-07-19T16:19:49Z DUMMY: affinity 0.9918806 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR 0.9999056 residue_name_number cleaner0 2023-07-19T18:10:06Z DUMMY: Val53β 0.9934963 complex_assembly cleaner0 2023-07-20T07:49:27Z GO: MHC 0.99990606 residue_name_number cleaner0 2023-07-19T18:10:12Z DUMMY: Gln72 0.9320513 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.9955964 complex_assembly cleaner0 2023-07-19T16:18:16Z GO: 1E6-A2-MVWGPDPLYV 0.9805279 complex_assembly cleaner0 2023-07-19T20:53:36Z GO: TCR RESULTS title_2 17164 An energetic switch from unfavorable to favorable entropy (order-to-disorder) correlates with antigen potency. evidence DUMMY: cleaner0 2023-07-19T16:19:43Z entropy RESULTS paragraph 17275 Our analysis of the contact network provided some clues that could explain the different antigen potencies and binding affinities between the 1E6 TCR and the different APLs. However, there were clear outliers in which the number of contacts did not match with the strength/potency of the interaction. For example, the 1E6 TCR bound to A2-RQWGPDPAAV with the third strongest affinity (KD = 7.8 μM) but made fewer contacts than with A2-ALWGPDPAAA (KD = ~208 μM) (Table 2). However, it is not necessarily the quantity of contacts that determines the strength of an interaction, but the quality of the contacts. Thus, we performed an in-depth thermodynamic analysis of 6 of the ligands under investigation (Figure 8 and Supplemental Table 3). The weak binding affinity between 1E6 and A2-MVWGPDPLYV and A2-YLGGPDFPTI generated thermodynamic data that were not robust enough to gain insight into the enthalpic (ΔH°) and entropic (TΔS°) changes that contributed to the different binding affinities/potencies for each APL. The overall free binding energies (ΔG°) were between –4.4 and –8.6 kcal/mol, reflecting the wide range of TCR binding affinities we observed for the different APLs. The enthalpic contribution in each complex did not follow a clear trend with affinity, with all but the 1E6-A2-RQFGPDFPTI interaction (ΔH° = 6.3 kcal/mol) generating an energetically favorable enthalpy value (ΔH° = –3.7 to –11.4 kcal/mol); this indicated a net gain in electrostatic interactions during complex formation. However, there was a clear switch in entropy between the weaker-affinity and stronger-affinity ligands, indicated by a strong Pearson’s correlation value between entropy and affinity (Pearson’s correlation value 0.93, P =0.007). For instance, the A2-ALWGPDPAAA, A2-AQWGPDAAA, and A2-RQFGPDWIVA (KD = ~208 μM, KD = 61.9 μM, and KD = 44.4 μM, respectively) were all entropically unfavorable (TΔS° = –2.9 to –5.6 kcal/mol), indicating a net change from disorder to order. Conversely, the stronger-affinity ligands A2-RQWGPDPAAV (KD = 7.8 μM), A2-YQFGPDFPIA (KD = 7.4 μM), and A2-RQFGPDFPTI (KD = 0.5 μM) exhibited favorable entropy (TΔS° = 2.2 to 14.9 kcal/mol), indicating an order-to-disorder change during binding, possibly through the expulsion of ordered water molecules. Furthermore, the structures of the unligated pMHCs demonstrated that, for these stronger-affinity ligands, there was less conformational difference between the TCR ligated pMHCs compared with the weaker-affinity ligands (Figure 6). The potential requirement for a larger degree of induced fit during binding to these weaker-affinity ligands is consistent with the larger entropic penalties observed for these interactions. 0.99799347 site cleaner0 2023-07-20T07:51:25Z SO: contact network 0.9993336 evidence cleaner0 2023-07-19T20:45:58Z DUMMY: binding affinities complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR chemical CHEBI: cleaner0 2023-07-19T16:29:10Z APLs complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9994259 protein_state cleaner0 2023-07-19T16:13:16Z DUMMY: bound to chemical CHEBI: cleaner0 2023-07-19T16:16:24Z A2-RQWGPDPAAV evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.9945314 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.67255753 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.99652267 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD experimental_method MESH: cleaner0 2023-07-20T08:07:10Z thermodynamic analysis 0.99823916 evidence cleaner0 2023-07-19T16:20:12Z DUMMY: binding affinity 0.92317814 chemical cleaner0 2023-07-19T16:12:10Z CHEBI: A2-MVWGPDPLYV 0.91059226 chemical cleaner0 2023-07-19T16:16:10Z CHEBI: A2-YLGGPDFPTI evidence DUMMY: cleaner0 2023-07-19T16:20:24Z enthalpic 0.9994355 evidence cleaner0 2023-07-19T20:46:04Z DUMMY: ΔH° evidence DUMMY: cleaner0 2023-07-19T16:20:29Z entropic 0.99951434 evidence cleaner0 2023-07-19T16:20:33Z DUMMY: TΔS° 0.9993646 evidence cleaner0 2023-07-19T20:46:07Z DUMMY: binding affinities chemical CHEBI: cleaner0 2023-07-19T16:29:16Z APL 0.99949455 evidence cleaner0 2023-07-19T20:46:12Z DUMMY: free binding energies 0.9995753 evidence cleaner0 2023-07-19T16:19:58Z DUMMY: ΔG° 0.9992965 evidence cleaner0 2023-07-19T18:10:40Z DUMMY: TCR binding affinities chemical CHEBI: cleaner0 2023-07-19T16:29:10Z APLs evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.99789983 complex_assembly cleaner0 2023-07-19T16:19:12Z GO: 1E6-A2-RQFGPDFPTI 0.99929667 evidence cleaner0 2023-07-19T20:46:15Z DUMMY: ΔH° 0.9959241 evidence cleaner0 2023-07-19T16:19:38Z DUMMY: enthalpy 0.9993475 evidence cleaner0 2023-07-19T16:19:34Z DUMMY: ΔH° evidence DUMMY: cleaner0 2023-07-19T16:19:43Z entropy evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.9310463 evidence cleaner0 2023-07-19T20:46:20Z DUMMY: Pearson’s correlation value 0.99341667 evidence cleaner0 2023-07-19T16:19:43Z DUMMY: entropy 0.9612975 evidence cleaner0 2023-07-19T16:19:48Z DUMMY: affinity 0.94222593 evidence cleaner0 2023-07-19T20:46:24Z DUMMY: Pearson’s correlation value evidence DUMMY: cleaner0 2023-07-19T20:46:32Z P 0.96640253 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.8315219 chemical cleaner0 2023-07-19T16:17:32Z CHEBI: A2-AQWGPDAAA 0.9568971 chemical cleaner0 2023-07-19T16:12:47Z CHEBI: A2-RQFGPDWIVA 0.9870682 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.968549 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.94423616 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.9992269 evidence cleaner0 2023-07-19T20:46:40Z DUMMY: TΔS° evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity chemical CHEBI: cleaner0 2023-07-19T16:16:24Z A2-RQWGPDPAAV 0.85928726 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD chemical CHEBI: cleaner0 2023-07-19T16:15:52Z A2-YQFGPDFPIA evidence DUMMY: cleaner0 2023-07-19T16:17:17Z KD chemical CHEBI: cleaner0 2023-07-19T16:12:27Z A2-RQFGPDFPTI evidence DUMMY: cleaner0 2023-07-19T16:17:17Z KD evidence DUMMY: cleaner0 2023-07-19T16:19:43Z entropy evidence DUMMY: cleaner0 2023-07-19T20:16:15Z TΔS° protein_state DUMMY: cleaner0 2023-07-19T16:17:41Z unligated complex_assembly GO: cleaner0 2023-07-19T16:28:11Z pMHCs evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity complex_assembly GO: cleaner0 2023-07-19T20:53:37Z TCR protein_state DUMMY: cleaner0 2023-07-19T20:05:05Z ligated complex_assembly GO: cleaner0 2023-07-19T16:28:11Z pMHCs evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity RESULTS title_2 20013 Potential epitopes for 1E6 TCR occur commonly in the viral proteome. complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.99776137 taxonomy_domain cleaner0 2023-07-19T20:08:47Z DUMMY: viral RESULTS paragraph 20082 We searched a database of over 1,924,572 unique decamer peptides from the proteome of viral pathogens that are known, or strongly suspected, to infect humans. Three hundred forty-two of these decamers conformed to the motif xxxGPDxxxx. Of these, 53 peptides contained the motif xOxGPDxxxO, where O is one of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 (Supplemental Table 4). Thus, there are many pathogen-encoded peptides that could act as agonists for the 1E6 T cell beyond the MVWGPDPLYV and RQFGPDWIVA sequences studied here. Extension of these analyses to include the larger genomes of bacterial pathogens would be expected to considerably increase these numbers. The binding affinity of the 1E6 TCR interaction with A2-RQFGPDWIVA is considerably higher than with the disease-implicated A2-ALWGPDPAAA sequence (KD = 44.4 μM and KD > 200 μM, respectively), highlighting how a pathogen-derived sequence might be capable of priming a 1E6-like T cell. 0.73867536 chemical cleaner0 2023-07-20T07:57:42Z CHEBI: peptides taxonomy_domain DUMMY: cleaner0 2023-07-19T20:08:49Z viral 0.9992586 species cleaner0 2023-07-19T20:43:40Z MESH: humans 0.9991959 structure_element cleaner0 2023-07-19T18:11:28Z SO: xxxGPDxxxx 0.99927825 structure_element cleaner0 2023-07-19T18:11:34Z SO: xOxGPDxxxO 0.99843377 residue_name cleaner0 2023-07-19T18:11:44Z SO: A 0.9987311 residue_name cleaner0 2023-07-19T18:11:47Z SO: V 0.99887043 residue_name cleaner0 2023-07-19T18:11:50Z SO: I 0.9988965 residue_name cleaner0 2023-07-19T18:11:53Z SO: L 0.998949 residue_name cleaner0 2023-07-19T18:11:56Z SO: M 0.999042 residue_name cleaner0 2023-07-19T18:11:58Z SO: Y 0.9989599 residue_name cleaner0 2023-07-19T18:12:01Z SO: F 0.99902046 residue_name cleaner0 2023-07-19T18:12:04Z SO: W 0.9695014 protein cleaner0 2023-07-19T20:43:55Z PR: HLA-A*0201 0.6446219 chemical cleaner0 2023-07-19T16:08:36Z CHEBI: MVWGPDPLYV 0.58860224 chemical cleaner0 2023-07-19T16:08:56Z CHEBI: RQFGPDWIVA 0.99951077 taxonomy_domain cleaner0 2023-07-19T20:08:55Z DUMMY: bacterial 0.99958223 evidence cleaner0 2023-07-19T16:20:13Z DUMMY: binding affinity complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.981877 chemical cleaner0 2023-07-19T16:12:47Z CHEBI: A2-RQFGPDWIVA 0.98185354 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.99964726 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.9996582 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD DISCUSS title_1 21094 Discussion DISCUSS paragraph 21105 T cell antigen discrimination is governed by an interaction between the clonally expressed TCR and pMHC, mediated by the chemical characteristics of the interacting molecules. It has recently become clear that TCR cross-reactivity with large numbers of different pMHC ligands is essential to plug holes in T cell immune coverage that pathogens could exploit. Flexibility at the interface between the TCR and pMHC, demonstrated in various studies, has been suggested as a mechanism mediating T cell cross-reactivity with multiple distinct epitopes. This notion is attractive because the CDR loops, which form the TCR antigen-binding site, are usually the most flexible part of the TCR and have the ability to mold around differently shaped ligands. Focused binding around a minimal peptide motif has also been implicated as an alternative mechanism enabling TCR cross-reactivity. Notably among these studies, Garcia and colleagues recently used the alloreactive murine TCR-MHC pair of the 42F3 TCR and H2-Ld to demonstrate recognition of a large number of different peptides via conserved hotspot contacts with prominent up-facing peptide residues. 0.9691974 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.93291223 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC 0.9701488 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.6247094 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC 0.99802315 site cleaner0 2023-07-20T08:13:53Z SO: interface 0.45847034 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.6944931 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC 0.9996326 structure_element cleaner0 2023-07-19T17:58:59Z SO: CDR loops 0.9995467 site cleaner0 2023-07-19T18:12:20Z SO: TCR antigen-binding site 0.98521197 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.9578315 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.8868667 protein_state cleaner0 2023-07-20T08:00:59Z DUMMY: alloreactive 0.9976369 taxonomy_domain cleaner0 2023-07-19T20:08:39Z DUMMY: murine 0.8430462 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR complex_assembly GO: cleaner0 2023-07-20T07:49:27Z MHC 0.71081364 protein cleaner0 2023-07-19T20:21:31Z PR: 42F3 0.7886316 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR protein PR: cleaner0 2023-07-19T20:18:16Z H2-Ld 0.9117572 protein_state cleaner0 2023-07-20T08:01:10Z DUMMY: conserved 0.996566 site cleaner0 2023-07-20T08:13:57Z SO: hotspot DISCUSS paragraph 22253 Sethi and colleagues recently demonstrated that the MHCII-restricted Hy.1B11 TCR, which was isolated from a patient with multiple sclerosis, could anchor into a deep pocket formed from peptide residues 2, 3, and 5 (from MBP85–99 bound to HLA-DQ1). This motif was conserved in at least 2 potential foreign peptides, originating from Herpes simplex virus and Pseudomonas aeruginosa, enabling TCR recognition of foreign epitopes. Although these data provided some clues into the molecular mechanism of T cell recognition, there still remain several gaps in our understanding. First, we currently know nothing about how human MHCI–restricted TCRs mediate cross-reactivity in the context of a clinically relevant model of autoimmunity, thought to be a major pathway of disease initiation in several autoimmune diseases. Second, molecular studies have not yet revealed a broad set of rules that determine TCR cross-reactivity because, with the exception of the allo–TCR-MHC pair of the 42F3 TCR and H2-Ld that did not encounter each other during T cell development, studies have been limited to structures of a TCR with only 2 or 3 different ligands. Finally, no studies have included characterization of pathogen-derived ligands recognized by self-reactive T cells with greater potency than the autoantigen, a potentially important facet to break self-tolerance. 0.35402265 protein_type cleaner0 2023-07-19T20:18:21Z MESH: MHCII protein PR: cleaner0 2023-07-19T20:20:30Z Hy.1B11 0.8917636 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.99890864 site cleaner0 2023-07-20T08:14:02Z SO: deep pocket 0.991907 residue_number cleaner0 2023-07-20T08:04:15Z DUMMY: 2 0.97971445 residue_number cleaner0 2023-07-20T08:04:18Z DUMMY: 3 0.9611459 residue_number cleaner0 2023-07-20T08:04:22Z DUMMY: 5 0.98578435 protein cleaner0 2023-07-19T20:20:51Z PR: MBP85–99 0.9992782 protein_state cleaner0 2023-07-19T16:13:16Z DUMMY: bound to 0.9985524 protein cleaner0 2023-07-19T20:43:59Z PR: HLA-DQ1 0.99849296 protein_state cleaner0 2023-07-19T20:21:23Z DUMMY: conserved 0.7983127 species cleaner0 2023-07-19T20:08:26Z MESH: Herpes simplex virus 0.9946457 species cleaner0 2023-07-19T20:43:47Z MESH: Pseudomonas aeruginosa 0.91344637 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.99882895 species cleaner0 2023-07-19T16:09:40Z MESH: human 0.5277726 complex_assembly cleaner0 2023-07-20T07:50:07Z GO: MHCI 0.93057656 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs 0.9568025 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR protein_state DUMMY: cleaner0 2023-07-20T08:03:53Z allo complex_assembly GO: cleaner0 2023-07-20T08:03:43Z TCR-MHC 0.9717623 protein cleaner0 2023-07-19T20:21:30Z PR: 42F3 0.6603948 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR protein PR: cleaner0 2023-07-19T20:18:27Z H2-Ld 0.99895006 evidence cleaner0 2023-07-19T20:46:46Z DUMMY: structures 0.98545176 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR DISCUSS paragraph 23617 Here, we investigated a highly cross-reactive MHCI-restricted TCR isolated from a patient with T1D that recognizes an HLA-A*0201–restricted preproinsulin signal peptide (ALWGPDPAAA15–24). Human CD8+ T cell clones expressing TCRs with this specificity mediate the destruction of β cells, have been found in islets early in infection, and are proposed to be a major driver of disease. We solved the structure of the 1E6 TCR with 7 APLs to enable a comprehensive analysis of the molecular basis of TCR degeneracy. The epitopes we selected exhibited a broad range of potencies and could activate the 1E6 T cell clone at exogenously supplied concentrations more than 4 logs apart. Overall, the difference in antigen potency correlated well with the binding energy (ΔG° kcal/mol) of the 1E6 TCR for the different epitopes, which ranged from values of ΔG° = ~–4.4 to –8.6 kcal/mol (calculated from 3D affinity data) or 2D affinity values of AcKa = 2.5 × 10–5 to 4.4 × 10–2 μm4. The weaker end of this spectrum extends our understanding of the limits in which T cells can functionally operate in terms of TCR 3D binding affinity and is in line with the types of very low affinity, yet fully functional self-reactive CD8+ T cells we have observed in tumor-infiltrating lymphocytes. Previous studies of autoreactive TCRs have shown that their binding mode is generally atypical, either due to an unusual binding manner, weak TCR binding affinity, an unstable pMHC, or a combination of these factors. Our data demonstrate the potential for an autoreactive TCR to bind with a conventional binding mode to a stable pMHC with antipathogen-like affinity (KD = 0.5 μM) depending on the peptide sequence. Our structural analysis revealed that the 1E6 TCR bound with a conserved conformation across all APLs investigated. This binding orientation was mediated through a focused interaction with TCR residues Tyr97α and Trp97β that formed an aromatic cap over a central ‘GDP’ motif that was common to all APLs. We have previously demonstrated the importance of the GPD motif using a peptide library scan, as well as a CPL scan approach. Although the 1E6 T cell was able to activate weakly with peptides that lacked this motif, we were unable to robustly measure binding affinities or generate complex structures with these ligands, highlighting the central role of this interaction during 1E6 T cell antigen recognition. This hotspot binding, defined as a localized cluster of interactions that dominate binding energy during protein-protein interactions, has been previously shown to contribute to TCR recognition of MHC as a mechanism that tunes T cell cross-reactivity by providing fixed anchor points that enable TCRs to tolerate a variable peptide cargo. Alternatively, interactions between the TCR and peptide have been shown to dominate the energetic landscape during ligand engagement, ensuring that T cells retain peptide specificity. The binding mechanism utilized by the 1E6 TCR during pMHC recognition is consistent with both of these models. Ligand engagement is dominated by peptide interactions, but hotspot-like interactions with the central GPD motif enable the 1E6 TCR to tolerate peptide residues that vary outside of this region, explaining how T cells expressing this TCR may cross-react with a large number of different peptides. These findings are also analogous to the observed binding mode of the Hy.1B11 TCR, in which one aromatic residue of the TCR CDR3α loop anchored into a pocket created by a conserved peptide motif. In both of these examples, self-recognition is mediated by TCR residues with aromatic side chains. These large, generally hydrophobic amino acids can form strong interactions with other residues through π-π stacking. Combined with evidence demonstrating that aromatic side chains are conserved in the CDR2 loops of TCRs from many species, we speculate that these aromatic residues could impart a level of “stickiness” to TCRs, which might be enriched in an autoimmune setting when the TCR often binds in a nonoptimal fashion. 0.81052303 complex_assembly cleaner0 2023-07-20T07:50:07Z GO: MHCI 0.979186 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR protein PR: cleaner0 2023-07-19T20:21:49Z HLA-A*0201 0.8616006 protein cleaner0 2023-07-19T20:09:35Z PR: preproinsulin structure_element SO: cleaner0 2023-07-20T07:58:57Z signal peptide chemical CHEBI: cleaner0 2023-07-19T18:13:27Z ALWGPDPAAA15–24 0.99920386 species cleaner0 2023-07-19T16:09:40Z MESH: Human 0.9869667 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs 0.9993399 experimental_method cleaner0 2023-07-20T07:52:01Z MESH: solved 0.9990907 evidence cleaner0 2023-07-19T20:46:51Z DUMMY: structure complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR chemical CHEBI: cleaner0 2023-07-19T16:29:10Z APLs 0.9110737 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.9994798 evidence cleaner0 2023-07-19T20:46:54Z DUMMY: binding energy 0.9995944 evidence cleaner0 2023-07-19T20:18:54Z DUMMY: ΔG° complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9996221 evidence cleaner0 2023-07-19T20:19:05Z DUMMY: ΔG° evidence DUMMY: cleaner0 2023-07-19T16:33:07Z 3D affinity evidence DUMMY: cleaner0 2023-07-19T16:32:22Z 2D affinity 0.9995473 evidence cleaner0 2023-07-19T20:46:58Z DUMMY: AcKa evidence DUMMY: cleaner0 2023-07-19T18:14:53Z TCR 3D binding affinity evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.9647545 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs 0.9988679 evidence cleaner0 2023-07-19T18:06:46Z DUMMY: TCR binding affinity 0.9996183 protein_state cleaner0 2023-07-20T08:01:18Z DUMMY: unstable 0.5334077 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC 0.93847495 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.99961215 protein_state cleaner0 2023-07-20T08:01:23Z DUMMY: stable 0.8652448 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity 0.9986419 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.99938667 experimental_method cleaner0 2023-07-19T20:19:26Z MESH: structural analysis complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.99946815 protein_state cleaner0 2023-07-20T08:01:45Z DUMMY: bound 0.9993304 protein_state cleaner0 2023-07-20T08:01:48Z DUMMY: conserved conformation 0.96806884 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.73016405 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.9998951 residue_name_number cleaner0 2023-07-19T18:00:26Z DUMMY: Tyr97α 0.99989617 residue_name_number cleaner0 2023-07-19T18:00:43Z DUMMY: Trp97β 0.984303 structure_element cleaner0 2023-07-20T08:14:14Z SO: aromatic cap structure_element SO: cleaner0 2023-07-20T08:10:19Z ‘GDP’ motif 0.97002655 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.9996266 structure_element cleaner0 2023-07-19T18:05:06Z SO: GPD motif 0.9995017 experimental_method cleaner0 2023-07-20T07:51:52Z MESH: peptide library scan 0.99955547 experimental_method cleaner0 2023-07-20T07:51:55Z MESH: CPL scan 0.9993992 protein_state cleaner0 2023-07-20T08:01:55Z DUMMY: lacked 0.9995267 evidence cleaner0 2023-07-19T20:47:04Z DUMMY: binding affinities 0.99743986 evidence cleaner0 2023-07-19T20:47:07Z DUMMY: structures complex_assembly GO: cleaner0 2023-07-19T20:53:37Z TCR complex_assembly GO: cleaner0 2023-07-20T07:49:27Z MHC complex_assembly GO: cleaner0 2023-07-19T20:55:24Z TCRs complex_assembly GO: cleaner0 2023-07-19T20:53:37Z TCR complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR complex_assembly GO: cleaner0 2023-07-19T16:24:26Z pMHC structure_element SO: cleaner0 2023-07-19T18:05:06Z GPD motif complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR complex_assembly GO: cleaner0 2023-07-19T20:53:37Z TCR protein PR: cleaner0 2023-07-19T20:20:31Z Hy.1B11 complex_assembly GO: cleaner0 2023-07-19T20:53:37Z TCR complex_assembly GO: cleaner0 2023-07-19T20:53:37Z TCR structure_element SO: cleaner0 2023-07-19T20:07:36Z CDR3α loop complex_assembly GO: cleaner0 2023-07-19T20:53:37Z TCR structure_element SO: cleaner0 2023-07-19T20:07:49Z CDR2 loops complex_assembly GO: cleaner0 2023-07-19T20:55:24Z TCRs complex_assembly GO: cleaner0 2023-07-19T20:55:24Z TCRs complex_assembly GO: cleaner0 2023-07-19T20:53:37Z TCR DISCUSS paragraph 27706 Despite some weak statistical correlation between the surface complementarity (SC) and affinity, closer inspection of the interface revealed no obvious structural signature that could definitively explain the differences in antigen potency and TCR binding strength between the different ligands. However, similar to our findings in other systems, modifications to residues outside of the canonical central peptide bulge were important for generating new interactions. For example, all of the stronger ligands encoded larger side chains (Arg or Tyr) at peptide position 1 that enabled new interactions with 1E6 not present with the Ala at this position in the natural preproinsulin peptide. These data also explain our previous findings that alteration of the anchor residue at peptide position 2 (Leu-Gln) has a direct effect on 1E6 TCR binding affinity because our structural analysis demonstrated that 1E6 made 3 additional bonds with A2-AQWGPDPAAA compared with A2-ALWGPDPAAA, consistent with the >3-fold stronger binding affinity. We have recently demonstrated how a suboptimal position 2 anchor in a melanoma-derived antigen can improve TCR binding through a similar mechanism. These results challenge the notion that the most potent peptide antigens exhibit the greatest pMHC stability and have implications for the design of anchor residue–modified heteroclitic peptides for vaccination. 0.9992144 evidence cleaner0 2023-07-19T20:19:30Z DUMMY: surface complementarity 0.9990908 evidence cleaner0 2023-07-19T20:19:34Z DUMMY: SC 0.95532745 evidence cleaner0 2023-07-19T16:19:49Z DUMMY: affinity 0.99941313 site cleaner0 2023-07-20T08:14:22Z SO: interface evidence DUMMY: cleaner0 2023-07-19T20:07:00Z antigen potency evidence DUMMY: cleaner0 2023-07-19T20:07:13Z TCR binding strength 0.9919825 structure_element cleaner0 2023-07-20T08:10:25Z SO: central peptide bulge 0.9993142 residue_name cleaner0 2023-07-19T18:16:23Z SO: Arg 0.9993111 residue_name cleaner0 2023-07-19T18:16:26Z SO: Tyr 0.8965547 residue_number cleaner0 2023-07-20T08:04:28Z DUMMY: 1 0.9992756 residue_name cleaner0 2023-07-19T18:16:28Z SO: Ala 0.80057234 protein cleaner0 2023-07-19T20:09:35Z PR: preproinsulin 0.7066019 chemical cleaner0 2023-07-20T07:59:05Z CHEBI: peptide 0.9753542 structure_element cleaner0 2023-07-20T08:10:30Z SO: anchor residue 0.79387724 residue_number cleaner0 2023-07-20T08:04:31Z DUMMY: 2 mutant MESH: cleaner0 2023-07-19T20:05:49Z Leu-Gln evidence DUMMY: cleaner0 2023-07-19T18:15:42Z 1E6 TCR binding affinity 0.99653417 experimental_method cleaner0 2023-07-20T08:07:18Z MESH: structural analysis 0.97112995 chemical cleaner0 2023-07-19T18:15:59Z CHEBI: A2-AQWGPDPAAA 0.97348905 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.99940276 evidence cleaner0 2023-07-19T16:20:13Z DUMMY: binding affinity residue_number DUMMY: cleaner0 2023-07-20T08:04:50Z 2 0.5003262 structure_element cleaner0 2023-07-20T08:10:33Z SO: anchor complex_assembly GO: cleaner0 2023-07-19T20:53:37Z TCR 0.62192553 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC DISCUSS paragraph 29103 Early thermodynamic analysis of TCR-pMHC interactions suggested a common energetic signature, driven by favorable enthalpy (generally mediated through an increase in electrostatic interactions) and unfavorable entropy (changes from disorder to order). These parameters aligned well with structural data, demonstrating that TCRs engaged pMHC using an induced fit binding mode. However, more recent data have shown that TCRs can utilize a range of energetic strategies during pMHC binding, currently with no obvious pattern in terms of TCR affinity, binding mechanism, or specificity (pathogen, cancer, or self-ligands). Although no energetic signature appears to exist for different TCRs, we used thermodynamic analysis here to explore whether changes in energetics could help explain ligand discrimination by a single TCR. This analysis demonstrated a strong relationship (according to the Pearson’s correlation analysis) between the energetic signature used by the 1E6 TCR and the sensitivity of the 1E6 T cell clone to different APLs. The weaker APL ligands were characterized by favorable enthalpy and unfavorable entropy, whereas the stronger ligands progressively shifted to favorable entropy. These differences were consistent with a greater degree of movement between the unligated and ligated pMHCs for the weaker ligands, suggesting a greater requirement for disorder-to-order changes during TCR binding. Thus, the enhanced antigen potency was probably mediated through a shift from an induced fit to a lock-and-key interaction between the stronger ligands (less requirement for energetically unfavorable disorder-to-order changes), resulting in a more energetically favorable ΔG value. 0.99953127 experimental_method cleaner0 2023-07-19T21:01:01Z MESH: thermodynamic analysis 0.9553528 complex_assembly cleaner0 2023-07-19T18:04:07Z GO: TCR-pMHC 0.9950689 evidence cleaner0 2023-07-19T16:19:39Z DUMMY: enthalpy 0.9896561 evidence cleaner0 2023-07-19T16:19:43Z DUMMY: entropy 0.9974977 evidence cleaner0 2023-07-19T20:47:13Z DUMMY: structural data 0.9933831 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs 0.7360548 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC 0.99634874 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs 0.5931357 complex_assembly cleaner0 2023-07-19T16:24:26Z GO: pMHC evidence DUMMY: cleaner0 2023-07-19T20:06:15Z TCR affinity 0.9940952 complex_assembly cleaner0 2023-07-19T20:55:24Z GO: TCRs 0.9995968 experimental_method cleaner0 2023-07-19T21:00:58Z MESH: thermodynamic analysis 0.99539065 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.9399544 experimental_method cleaner0 2023-07-19T16:31:19Z MESH: Pearson’s correlation analysis complex_assembly GO: cleaner0 2023-07-19T20:54:51Z 1E6 TCR 0.9980301 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.99799025 chemical cleaner0 2023-07-19T16:29:16Z CHEBI: APL 0.9958858 evidence cleaner0 2023-07-19T16:19:39Z DUMMY: enthalpy 0.9963213 evidence cleaner0 2023-07-19T16:19:43Z DUMMY: entropy 0.9973941 evidence cleaner0 2023-07-19T16:19:43Z DUMMY: entropy 0.9996511 protein_state cleaner0 2023-07-19T16:17:41Z DUMMY: unligated 0.99963677 protein_state cleaner0 2023-07-19T20:05:05Z DUMMY: ligated 0.6019646 complex_assembly cleaner0 2023-07-19T16:28:11Z GO: pMHCs 0.85927975 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.9993516 evidence cleaner0 2023-07-19T20:23:20Z DUMMY: ΔG value DISCUSS paragraph 30803 Importantly, the preproinsulin-derived epitope was one of the least potent peptides, demonstrating that the 1E6 T cell clone had the ability to respond to different peptide sequences with far greater potency. The RQFGPDWIVA peptide, which was substantially more potent than the preproinsulin peptide, is within the proteome of a common human pathogen (C. asparagiforme), demonstrating the potential for an encounter between a naive 1E6-like T cell and a foreign peptide with a more potent ligand that might then break self-tolerance. Indeed, we found over 50 decamer peptides from the proteome of likely, or known, human viral pathogens alone that contained both the conserved central GPD motif and anchor residues at positions 2 and 10 that would enable binding to HLA-A*02:01. Further experiments will be required to determine whether any naturally presented, human pathogen–derived peptides act as active ligands for 1E6, but our work presented here demonstrates that it is at least feasible for an autoimmune TCR to bind to a different peptide sequence that could be present in a pathogen proteome with substantially higher affinity and potency than the interaction it might use to attack self-tissue. 0.28811467 protein cleaner0 2023-07-19T20:09:35Z PR: preproinsulin 0.3508806 chemical cleaner0 2023-07-19T16:08:56Z CHEBI: RQFGPDWIVA 0.3288183 protein cleaner0 2023-07-19T20:09:35Z PR: preproinsulin 0.99934274 species cleaner0 2023-07-19T16:09:40Z MESH: human 0.9992831 species cleaner0 2023-07-19T16:09:34Z MESH: C. asparagiforme 0.99928576 species cleaner0 2023-07-19T16:09:40Z MESH: human taxonomy_domain DUMMY: cleaner0 2023-07-19T20:08:49Z viral 0.99951875 protein_state cleaner0 2023-07-20T08:02:00Z DUMMY: conserved 0.99971485 structure_element cleaner0 2023-07-19T18:05:06Z SO: GPD motif 0.9981116 structure_element cleaner0 2023-07-20T08:10:39Z SO: anchor residues 0.99485046 residue_number cleaner0 2023-07-20T08:04:56Z DUMMY: 2 0.9937171 residue_number cleaner0 2023-07-20T08:04:58Z DUMMY: 10 protein PR: cleaner0 2023-07-19T20:23:42Z HLA-A*02:01 0.9993616 species cleaner0 2023-07-19T16:09:40Z MESH: human 0.9938782 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR evidence DUMMY: cleaner0 2023-07-19T16:19:49Z affinity DISCUSS paragraph 32011 In summary, this investigation into the molecular basis of T cell cross-reactivity using a clinically relevant cytotoxic CD8+ T cell clone that kills human pancreatic β cells provides answers to a number of previously outstanding questions. First, our data shows that a single TCR has the potential to functionally (assessed through T cell activation) bind to different ligands with affinities ranging across 3 orders of magnitude. Second, this is the first example in which ligands have been identified and characterized for a human autoreactive TCR that are substantially more potent than the natural self-ligand, demonstrating the potential for a pathogenic ligand to break self-tolerance and prime self-reactive T cells. Third, this first structural analysis of a cross-reactive human MHCI–restricted autoimmune TCR showed that degeneracy was mediated through TCR-pMHC anchoring by a conserved minimal binding peptide motif. Finally, TCR ligand discrimination was characterized by an energetic shift from an enthalpically to entropically driven interaction. Our demonstration of the molecular mechanism governing cross-reactivity by this preproinsulin reactive human CD8+ T cell clone supports the notion first put forward by Wucherpfennig and Strominger that molecular mimicry could mediate autoimmunity and has far-reaching implications for the complex nature of T cell antigen discrimination. 0.99927574 species cleaner0 2023-07-19T16:09:40Z MESH: human 0.99455017 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.9996008 evidence cleaner0 2023-07-19T20:47:18Z DUMMY: affinities 0.9992525 species cleaner0 2023-07-19T16:09:40Z MESH: human 0.99530643 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.9987658 experimental_method cleaner0 2023-07-19T20:24:12Z MESH: structural analysis 0.9992393 species cleaner0 2023-07-19T16:09:40Z MESH: human complex_assembly GO: cleaner0 2023-07-20T07:50:07Z MHCI 0.9947507 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR complex_assembly GO: cleaner0 2023-07-19T18:04:07Z TCR-pMHC 0.99901617 protein_state cleaner0 2023-07-20T08:02:04Z DUMMY: conserved 0.9989063 structure_element cleaner0 2023-07-20T08:10:43Z SO: minimal binding peptide motif 0.9823903 complex_assembly cleaner0 2023-07-19T20:53:37Z GO: TCR 0.99480104 protein cleaner0 2023-07-19T20:09:35Z PR: preproinsulin 0.9992594 species cleaner0 2023-07-19T16:09:40Z MESH: human METHODS title_1 33414 Methods METHODS title_2 33422 T cell maintenance and culture. METHODS paragraph 33454 The 1E6 T cell clone was generated as previously described and stored in vapor phase liquid nitrogen in freezing buffer (90% FCS and 10% DMSO). Cells were defrosted rapidly in a 37°C water bath until a small amount of frozen cells were left and then immediately washed in 15–20 ml of R10 media (RPMI 1640 with 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine) by centrifuging at 300 g for 5 minutes. Defrosted cells were cultured in T cell media: R10 with 1× nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES buffer (all from Invitrogen), 20 IU/ml of IL-2 (aldesleukin, brand name Proleukin, Prometheus) and 25 ng/ml IL-15 (PeproTech), for 24 hours; then, 0.75 × 106 to 1.5 × 106 cells expanded by coculture with 15 × 106 irradiated (3,100 cGy) PBMCs from 3 donors in a 25 cm2 tissue culture flask with 1 μg/ml of phytoheamagglutinin (Alere Inc.) and T cell media as above. The clone was transferred to 24-well tissue culture plates (3 × 106 to 4 × 106 per well in 2 ml) at day 7, and the IL-2 increased to 200 IU/ml. For the purpose of this study, the clone was passaged 3 times and used between weeks 2 and 4 after expansion. METHODS title_2 34626 T cell activation assays and tetramer staining. METHODS paragraph 34674 The [51Cr] release cytotoxicity assay was performed as previously described. Target A2 CIR cells were labeled for 1 hour at 37°C with 30 μCi chromium (sodium chromate in normal saline, PerkinElmer) per 1 × 106 cells, washed with R10, and allowed to leach for a further hour at 37°C in R10 to remove any excess chromium from the cells. After chromium labeling, target cells were washed and plated at 1,000 cells/well in 96-well tissue culture plates and pulsed with peptide at the indicated concentrations for 2 hours at 37°C. T cells were added to give the desired T cell/target cell (5:1) ratio and a final volume of 150 μl R10. Target cells were also incubated alone or with 1% Triton X-100 detergent (Sigma-Aldrich) to give the spontaneous and total chromium released from the target cells, respectively. After overnight incubation, at 37°C and 5% CO2, the supernatants were both (i) assayed for MIP1β by ELISA (R&D Systems) and (ii) harvested (10% of total volume), mixed with 150 μl Optipahse supermix scintillation mixture (PerkinElmer) in 96-well polyethylene terephthalate plates (PerkinElmer), and sealed; the amount of released chromium was measured indirectly on a 1450 Microbeta counter (PerkinElmer). The percentage of specific target cell lysis by T cells was calculated according to the following formula: (experimental release [with T cells and target cells] − spontaneous release from target cells)/(total release from target cells − spontaneous release from target cells) × 100. Experiments were independently completed in triplicate. Tetrameric pMHCI reagents (tetramers) were constructed by the addition of PE-conjugated streptavidin (Invitrogen) at a pMHCI/streptavidin molar ratio of 4:1. A total of 50,000 T cells were stained with PE-conjugated tetramer (25 μg/ml) folded around the indicated peptides for 30 minutes on ice and washed with PBS before staining with 2 μl (1:40 dilution of the DMSO stock in PBS) of the violet LIVE/DEAD fixable dead cell stain Vivid (Invitrogen) for 5 minutes at room temperature before direct addition of 2 μl of anti–CD8-APC antibody (clone BW135/80, Miltenyi Biotec) and incubated for a further 20 minutes on ice before being washed in FACS buffer (2% FCS in PBS). Data were acquired using a FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc.). METHODS title_2 37041 Protein expression, refolding, and purification. METHODS paragraph 37090 The 1E6 TCR, HLA-A*0201, and human β2m chain were generated as described previously. The 1E6 TCR and HLA-A*0201 peptide variants were refolded and purified as described previously. Biotinylated pMHCI and pMHC tetramer were prepared as previously described. METHODS title_2 37350 pMHC stability assays. METHODS paragraph 37373 Thermal stability of the HLA-A*0201–peptide complexes was assessed by circular dichroism (CD) spectroscopy monitoring the change in ellipticities at 218 nm. Data were collected, in duplicate, using a nitrogen-flushed Module B end-station spectrophotometer at the B23 Synchrotron Radiation CD Beamline at the Diamond Light Source (DLS). Samples were prepared in phosphate buffered saline, pH 7.4, and concentrated to ~10 mM. Spectra were measured every 5°C over a temperature range between 5°C and 90°C with 5 minutes of equilibration time for each temperature. Four scans were acquired using an integration time of 1 second, a path length of 0.02 cm, and a slit width of 0.5 mm equivalent to a 1.2-nm bandwidth. Reversibility was monitored by measuring the spectrum at 20°C after cooling from 90°C with 30 minutes of incubation. Melting curves were analyzed assuming a 2-state trimer-to-monomer transition from the native (N) to unfolded (U) conformation N3 ↔ 3U with an equilibrium constant K = (U)3/(N3) = F/(3c2[1-F]3), where F and c are the degree of folding and protein concentration, respectively. Data were fitted as described. Fitted parameters were the melting temperature Tm, van’t Hoff’s enthalpy ΔHvH, and the slope and intercept of the native baseline. As all protein complexes aggregated to various degrees upon unfolding, the ellipticity of the unfolded state was set as a constant of –4,500 deg cm2/dmol. METHODS title_2 38810 SPR analysis. METHODS paragraph 38824 Binding analysis was performed using a BIAcore T200 equipped with a CM5 sensor chip as previously described. Binding analysis was performed 3× in independent experiments using pMHC monomers generated in-house. Approximately 200–500 RU of each HLA-A*0201–peptide complex was attached to the CM5 sensor chip at a slow flow rate of 10 μl/min to ensure uniform distribution on the chip surface. The 1E6 TCR was purified and concentrated to approximately 40–350 μM on the same day of SPR analysis to reduce the likelihood of TCR aggregation. For equilibrium analysis, 10 serial dilutions were prepared in triplicate for each sample and injected over the relevant sensor chips at 25°C. TCR was injected over the chip surface using kinetic injections at a flow rate of 45 μl/min using HLA-A*0201–ELAGIGILTV as a negative control surface on flow cell 1. For the thermodynamics experiments, this method was repeated at the following temperatures: 5°C, 13°C, 18°C, 25°C, 30°C, and 37°C. Results were analyzed using BIAevaluation 3.1, Excel, and Origin 6.0 software. The KD values were calculated assuming a 1:1 interaction by plotting specific equilibrium-binding responses against protein concentrations, followed by nonlinear least squares fitting of the Langmuir binding equation. The thermodynamic parameters were calculated using the nonlinear van’t Hoff equation (RT ln KD = ΔH° –TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0]) with T0=298 K. METHODS title_2 40285 Adhesion frequency assay. METHODS paragraph 40311 We used an adhesion frequency assay to measure the 2D affinity of TCR-pMHC interactions at the cell membrane as previously described. Briefly, human 1E6 T cells were mounted onto 1 micropipette, and, on the other pipette, human rbcs coated with pMHC by biotin-streptavidin coupling served as both a surrogate APC and an adhesion sensor for detecting the TCR-pMHC interaction. Site densities of TCR and pMHC were measured by flow cytometry as previously described. All assays were performed using at least 5 cell pairs and calculated as an average of 100 cell-cell contacts. METHODS title_2 40885 Crystal structure determination. METHODS paragraph 40918 All protein crystals were grown at 18°C by vapor diffusion via the sitting drop technique. Each pMHCI (200 nl, 10 mg/ml) in crystallization buffer (10 mM TRIS [pH 8.1] and 10 mM NaCl) was added to 200 nl of reservoir solution. HLA-A*0201–MVWGPDPLYV (A2-MVW) crystals were grown in 0.2 M ammonium chloride, 0.1 M TRIS (pH 8), 20% PEG 6000; HLA-A*0201–YLGGPDFPTI (A2-YLG) crystals were grown in 0.2 M sodium nitrate, 0.1 M BIS TRIS propane (pH 6.5), 20% PEG 3350; HLA-A*0201–AQWGPDPAAA (A2-AQW) crystals were grown in 0.2 M sodium malonate, 0.1 M BIS TRIS propane (pH 6.5), 20% PEG3350; HLA-A*0201–RQFGPDWIVA (A2-RQF[A]) crystals were grown in 0.2 M sodium sulphate, 0.1 M BIS TRIS propane (pH 6.5), 20% PEG 3350; HLA-A*0201–RQWGPDPAAV (A2-RQW) crystals were grown in 0.1 M TRIS (pH 8), 20% PEG 8000, 15% glycerol; HLA-A*0201–YQFGPDFPTA (A2-YQF) crystals were grown in 0.1 M TRIS (pH 8), 25% PEG 4000, 15% glycerol; HLA-A*0201–RQFGPDFPTI (A2-RQF[I]) crystals were grown in 0.2 M potassium/sodium tartrate, 0.1 M BIS TRIS propane (pH 8.5), 20% PEG 3350; 1E6-A2-MVW crystals were grown in 0.1 M HEPES (pH 7.5), 15% PEG 4000, 0.2 M sodium acetate; 1E6-A2-YLG crystals were grown in 0.1 M sodium cacodylate (pH 6.5), 15% PEG 4000, 0.2 M sodium acetate; 1E6-A2-AQW crystals were grown in 0.2 M sodium citrate, 0.1 M BIS TRIS propane (pH 6.5), 20% PEG 3350; 1E6-A2-RQF(A) crystals were grown in 0.1 M HEPES (pH 7), 15% PEG 4000, 0.2 M sodium acetate; 1E6-A2-RQW crystals were grown in 0.2 M sodium cholride, 0.1 M MES (pH 6), 20% PEG 6000; 1E6-A2-YQF crystals were grown in 0.2 M sodium cholride, 0.1 M HEPES (pH 7), 20% PEG 3350; and 1E6-A2-RQF(I) crystals were grown in 0.1 M HEPES (pH 7.5), 15% PEG 4000, 0.2 M sodium acetate. Crystallization screens were conducted using an Art-Robbins Phoenix dispensing robot (Alpha Biotech Ltd.), and data were collected at 100 K at the DLS at a wavelength of 0.98 Å using an ADSC Q315 CCD detector. Reflection intensities were estimated using XIA2, and the data were analyzed with Scala and the CCP4 package. Structures were solved with molecular replacement using Phaser. Sequences were adjusted with Coot, and the models were refined with REFMAC5. Graphical representations were prepared with PyMOL. The reflection data and final model coordinates were deposited with the PDB database (A2-MVW PDB: 5C0H; A2-YLG PDB: 5C0G; A2-AQW PDB: 5C0D; A2-RQF[A] PDB: 5C0J; A2-RQW PDB: 5C0F; A2-YQF PDB: 5C0E; A2-RQF[I] PDB: 5C0I; 1E6-A2-MVW PDB: 5C0A; 1E6-A2-YLG PDB: 5C09; 1E6-A2-AQW PDB: 5HYJ; 1E6-A2-RQF[A] PDB: 5C0C; 1E6-A2-RQW PDB: 5C08; 1E6-A2-YQF PDB: 5C07; and 1E6-A2-RQF[I] PDB: 5C0B). METHODS title_2 43552 Peptide motif predictions. METHODS paragraph 43579 Peptide motif predictions were performed by searching a viral database compiled using publicly available protein sequences of over 1,924,572 unique decamer peptides from the proteome of viral pathogens. The motif xOxGPDxxxO — where O is anyone of the hydrophobic amino acid residues A,V, I, L, M, Y, F, and W that might allow binding to HLA-A*0201 — was used as the search parameter. METHODS title_2 43967 Statistics. METHODS paragraph 43979 Pearson’s correlation analysis was performed to determine the relationship between TCR binding affinity and antigen potency, structural correlates, or thermodynamics using Origin Lab 9.0 pro. AUTH_CONT title_1 44173 Author contributions AUTH_CONT paragraph 44194 AMB, GD, AJS, BS, WR, AT, PJ, AF, AS, JJM, LW, PJR, and DKC performed experiments and analyzed the data. AKS, JR, CZ, JJM, MP, and DKC wrote the manuscript. AKS and DKC conceived and directed the study. AKS and DKC funded the study. All authors contributed to discussions. SUPPL title_1 44467 Supplementary Material SUPPL footnote 44490 Conflict of interest: The authors have declared that no conflict of interest exists. SUPPL footnote 44575 License: This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. SUPPL footnote 44755 Reference information:J Clin Invest. 2016;126(6):2191–2204. doi:10.1172/JCI85679. 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REF ref 52248 [published online ahead of print February 5, 2016]. doi:10.1038/icb.2016.12 surname:Szomolay;given-names:B 10.1038/icb.2016.12 26846725 REF Immunol Cell Biol ref 52249 Identification of human viral protein-derived ligands recognized by individual major histocompatibility complex class I (MHCI)-restricted T-cell receptors JCI85679.f1.jpg F1 FIG fig_title_caption 52404 The 1E6 T cell clone reacts with a broad sensitivity range to APLs. 0.9845797 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs JCI85679.f1.jpg F1 FIG fig_caption 52472 (A and B) The 1E6 T cell clone was tested in a peptide dilution assay, in triplicate, with MVWGPDPLYV (gray), YLGGPDFPTI (red), ALWGPDPAAA (blue), AQWGPDPAAA (green), RQFGPDWIVA (dark blue), RQWGPDPAAV (purple), YQFGPDFPTA (yellow), and RQFGPDFPTI (cyan) peptides presented by HLA-A*0201–expressing C1R cells for release of MIP-1β (A) and killing (B). (C) The 1E6 T cell clone was stained, in duplicate, with tetramers composed of each APL (colored as above) presented by HLA-A*0201. (D) The stability of each APL (colored as above) was tested, in duplicate, using CD by recording the peak at 218 nm absorbance from 5°C–90°C. Tm values were calculated using a Boltzmann fit to each set of data. 0.999573 experimental_method cleaner0 2023-07-19T20:25:01Z MESH: peptide dilution assay 0.9895058 chemical cleaner0 2023-07-19T16:08:36Z CHEBI: MVWGPDPLYV 0.9752729 chemical cleaner0 2023-07-19T16:09:11Z CHEBI: YLGGPDFPTI 0.9628796 chemical cleaner0 2023-07-19T16:08:20Z CHEBI: ALWGPDPAAA 0.96822727 chemical cleaner0 2023-07-19T18:27:26Z CHEBI: AQWGPDPAAA 0.99096864 chemical cleaner0 2023-07-19T16:08:56Z CHEBI: RQFGPDWIVA 0.99126 chemical cleaner0 2023-07-19T18:27:35Z CHEBI: RQWGPDPAAV 0.986967 chemical cleaner0 2023-07-19T18:27:44Z CHEBI: YQFGPDFPTA 0.98250383 chemical cleaner0 2023-07-19T18:27:52Z CHEBI: RQFGPDFPTI 0.89255476 protein cleaner0 2023-07-19T20:44:04Z PR: HLA-A*0201 0.8122008 protein cleaner0 2023-07-19T20:12:01Z PR: MIP-1β 0.9930211 oligomeric_state cleaner0 2023-07-20T07:58:34Z DUMMY: tetramers 0.9965977 chemical cleaner0 2023-07-19T16:29:17Z CHEBI: APL 0.99024296 protein cleaner0 2023-07-19T20:44:08Z PR: HLA-A*0201 0.997544 chemical cleaner0 2023-07-19T16:29:17Z CHEBI: APL 0.99962974 experimental_method cleaner0 2023-07-19T20:24:56Z MESH: CD 0.99954647 evidence cleaner0 2023-07-19T16:29:58Z DUMMY: Tm experimental_method MESH: cleaner0 2023-07-19T18:28:20Z Boltzmann fit to each set of data JCI85679.f2.jpg F2 FIG fig_title_caption 53174 3D and 2D binding analysis of the 1E6 TCR with A2-ALW and the APLs. experimental_method MESH: cleaner0 2023-07-20T08:07:41Z 3D and 2D binding analysis complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.8336919 chemical cleaner0 2023-07-19T20:41:38Z CHEBI: A2-ALW 0.5477331 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs JCI85679.f2.jpg F2 FIG fig_caption 53242 (A–H) Binding affinity of the 1E6 TCR interaction at 25°C using SPR. Eight serial dilutions of the 1E6 TCR were measured (shown in the inset); representative data from 3 independent experiments are plotted. The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y= [P1x]/[P2 + X]). In order to calculate each response, the 1E6 TCR was also injected over a control sample (HLA-A*0201–ILAKFLHWL) that was deducted from the experimental data. (A) 1E6-A2-MVWGPDPLYV (approximate value); (B) 1E6-A2-YLGGPDFPTI (approximate value); (C) 1E6-A2-ALWGPDPAAA; (D) 1E6-A2-AQWGPDPAAA; (E) 1E6-A2-RQFGPDWIVA; (F) 1E6-A2-RQWGPDPAAV; (G) 1E6-A2-YQFGPDFPTA; and (H) 1E6-A2-RQFGPDFPTI. (I) ΔG values, calculated from SPR experiments, plotted against 1/EC50 (the reciprocal peptide concentration required to reach half-maximal 1E6 T cell killing) showing Pearson’s coefficient analysis (r) and P value (including approximate values from A and B). (J) Effective 2D affinity (AcKa) calculated using adhesion frequency assays, using at least 5 cell pairs, and calculated as an average of 100 cell cell contacts. (K) Effective 2D affinity plotted against 1/EC50 showing Pearson’s coefficient analysis (r) and P value. 0.9995432 evidence cleaner0 2023-07-19T16:20:13Z DUMMY: Binding affinity complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.99968934 experimental_method cleaner0 2023-07-19T16:32:30Z MESH: SPR complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.99949384 evidence cleaner0 2023-07-19T18:28:45Z DUMMY: equilibrium binding constant 0.9995623 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.9307487 experimental_method cleaner0 2023-07-19T21:06:13Z MESH: nonlinear curve fit complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR complex_assembly GO: cleaner0 2023-07-19T20:42:26Z HLA-A*0201–ILAKFLHWL 0.9859878 complex_assembly cleaner0 2023-07-19T16:18:17Z GO: 1E6-A2-MVWGPDPLYV complex_assembly GO: cleaner0 2023-07-19T18:29:39Z 1E6-A2-YLGGPDFPTI 0.9805851 complex_assembly cleaner0 2023-07-19T16:14:43Z GO: 1E6-A2-ALWGPDPAAA 0.9903617 complex_assembly cleaner0 2023-07-19T18:30:04Z GO: 1E6-A2-AQWGPDPAAA 0.9882841 complex_assembly cleaner0 2023-07-19T18:30:24Z GO: 1E6-A2-RQFGPDWIVA 0.9882075 complex_assembly cleaner0 2023-07-19T18:30:45Z GO: 1E6-A2-RQWGPDPAAV 0.9757681 complex_assembly cleaner0 2023-07-19T18:29:50Z GO: 1E6-A2-YQFGPDFPTA 0.98563766 complex_assembly cleaner0 2023-07-19T16:19:13Z GO: 1E6-A2-RQFGPDFPTI 0.9887948 evidence cleaner0 2023-07-19T20:25:35Z DUMMY: ΔG values 0.9996978 experimental_method cleaner0 2023-07-19T16:32:30Z MESH: SPR evidence DUMMY: cleaner0 2023-07-19T20:42:36Z EC50 0.9450487 experimental_method cleaner0 2023-07-20T08:07:47Z MESH: Pearson’s coefficient analysis 0.84214836 evidence cleaner0 2023-07-19T20:25:14Z DUMMY: r 0.7447264 evidence cleaner0 2023-07-19T20:25:17Z DUMMY: P 0.99866694 evidence cleaner0 2023-07-19T20:47:26Z DUMMY: Effective 2D affinity 0.99921787 evidence cleaner0 2023-07-19T20:47:31Z DUMMY: AcKa 0.99958515 experimental_method cleaner0 2023-07-20T08:07:51Z MESH: adhesion frequency assays 0.9983494 evidence cleaner0 2023-07-19T20:47:35Z DUMMY: Effective 2D affinity 0.5637522 evidence cleaner0 2023-07-19T20:47:38Z DUMMY: EC50 0.97026414 experimental_method cleaner0 2023-07-20T08:07:54Z MESH: Pearson’s coefficient analysis 0.8508054 evidence cleaner0 2023-07-19T20:25:20Z DUMMY: r 0.8390739 evidence cleaner0 2023-07-19T20:25:26Z DUMMY: P value JCI85679.f3.jpg F3 FIG fig_title_caption 54482 The 1E6 TCR uses a conserved binding mode to engage A2-ALWGPDPAAA and the APLs. complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9342026 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.99728084 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs JCI85679.f3.jpg F3 FIG fig_caption 54562 (A) Superposition of the 1E6 TCR (multicolored illustration) in complex with all 7 APLs (multicolored sticks) and the A2-ALWGPDPAAA ligand using the HLA-A*0201 (gray illustration) molecule to align all of the structures. The 1E6 TCR and each peptide are colored according to the APL used in the complex as in Figure 1. (B) Position of the 1E6 TCR CDR loops (multicolored lines) in each complex. The ALWGPDPAAA peptide (green sticks) is shown in the HLA-A*0201 binding groove (gray surface). (C) The Cα backbone conformation of each APL (multicolored illustration) in the context of the HLA-A*0201 α1 helices (gray illustration). (D) Crossing angle of the 1E6 TCR (multicolored lines) calculated using previously published parameters in the context of the ALWGPDPAAA peptide (green sticks) bound in the HLA-A*0201 binding groove (gray surface). 0.999629 experimental_method cleaner0 2023-07-20T08:07:59Z MESH: Superposition complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR protein_state DUMMY: cleaner0 2023-07-19T18:02:50Z in complex with 0.8016965 chemical cleaner0 2023-07-19T16:29:10Z CHEBI: APLs 0.6889396 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.9861517 protein cleaner0 2023-07-19T20:44:14Z PR: HLA-A*0201 0.66666234 experimental_method cleaner0 2023-07-19T21:06:42Z MESH: align 0.9962159 evidence cleaner0 2023-07-19T20:47:43Z DUMMY: structures complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR chemical CHEBI: cleaner0 2023-07-19T16:29:17Z APL complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9996439 structure_element cleaner0 2023-07-19T17:58:59Z SO: CDR loops 0.99761826 chemical cleaner0 2023-07-19T16:08:20Z CHEBI: ALWGPDPAAA site SO: cleaner0 2023-07-20T08:08:20Z HLA-A*0201 binding groove 0.9049958 chemical cleaner0 2023-07-19T16:29:17Z CHEBI: APL 0.9774972 protein cleaner0 2023-07-19T20:44:17Z PR: HLA-A*0201 0.9996949 structure_element cleaner0 2023-07-20T08:10:49Z SO: α1 helices 0.9993441 evidence cleaner0 2023-07-19T20:47:49Z DUMMY: Crossing angle complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9979407 chemical cleaner0 2023-07-19T16:08:20Z CHEBI: ALWGPDPAAA 0.99416566 protein_state cleaner0 2023-07-20T08:02:23Z DUMMY: bound in site SO: cleaner0 2023-07-19T20:43:29Z HLA-A*0201 binding groove JCI85679.f4.jpg F4 FIG fig_title_caption 55414 A conserved interaction with a GPD motif underpins the 1E6 TCR interaction with the APLs. 0.9997474 structure_element cleaner0 2023-07-19T18:05:06Z SO: GPD motif complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.94330746 chemical cleaner0 2023-07-19T16:29:11Z CHEBI: APLs JCI85679.f4.jpg F4 FIG fig_caption 55504 Interaction between 1E6 TCR (gray illustration) residues Tyr97α and Tyr97β (the position of these side chains in the TCR in complex with all 7 APLs, and the previously reported A2-ALWGPDPAAA epitope, is shown in multicolored sticks; ref.) and the GPD peptide motif (the position of these side chains in all 7 APLs and A2-ALWGPDPAAA in complex with the 1E6 TCR is shown in multicolored sticks). The rest of the peptide, and the MHCα1 helix, are shown as a gray illustration. complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.99990344 residue_name_number cleaner0 2023-07-19T18:00:26Z DUMMY: Tyr97α 0.9999007 residue_name_number cleaner0 2023-07-19T20:04:48Z DUMMY: Tyr97β 0.63116497 complex_assembly cleaner0 2023-07-19T20:53:38Z GO: TCR protein_state DUMMY: cleaner0 2023-07-19T18:02:50Z in complex with chemical CHEBI: cleaner0 2023-07-19T16:29:11Z APLs 0.99554044 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA structure_element SO: cleaner0 2023-07-19T21:07:22Z GPD peptide motif chemical CHEBI: cleaner0 2023-07-19T16:29:11Z APLs 0.99627393 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.98936254 protein_state cleaner0 2023-07-19T18:02:50Z DUMMY: in complex with complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR complex_assembly GO: cleaner0 2023-07-20T08:11:09Z MHC structure_element SO: cleaner0 2023-07-20T08:11:19Z α1 helix JCI85679.f5.jpg F5 FIG fig_title_caption 55989 The 1E6 TCR makes distinct peptide contacts with peripheral APL residues. complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9978986 chemical cleaner0 2023-07-19T16:29:17Z CHEBI: APL JCI85679.f5.jpg F5 FIG fig_caption 56063 Interactions between the 1E6 TCR and peptide residues outside of the conserved GPD motif. The MHCα1 helix is shown in gray illustrations. Hydrogen bonds are shown as red dotted lines; van der Waals (vdW) contacts are shown as black dotted lines. Boxes show total contacts between the 1E6 TCR and each peptide ligand. (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.99947935 protein_state cleaner0 2023-07-20T08:02:45Z DUMMY: conserved 0.9997277 structure_element cleaner0 2023-07-19T18:05:06Z SO: GPD motif complex_assembly GO: cleaner0 2023-07-20T08:03:02Z MHC structure_element SO: cleaner0 2023-07-20T08:03:13Z α1 helix bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:18:18Z Hydrogen bonds bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:18:18Z van der Waals (vdW) contacts complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9976714 chemical cleaner0 2023-07-19T16:12:10Z CHEBI: A2-MVWGPDPLYV complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.99183035 chemical cleaner0 2023-07-19T16:16:11Z CHEBI: A2-YLGGPDFPTI complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9978401 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.99737835 chemical cleaner0 2023-07-19T18:34:05Z CHEBI: A2-AQWGPDPAAA complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.99795914 chemical cleaner0 2023-07-19T16:12:48Z CHEBI: A2-RQFGPDWIVA complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9971487 chemical cleaner0 2023-07-19T16:16:24Z CHEBI: A2-RQWGPDPAAV complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.99718714 chemical cleaner0 2023-07-19T18:33:55Z CHEBI: A2-YQFGPDFPTA complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9949441 chemical cleaner0 2023-07-19T16:12:27Z CHEBI: A2-RQFGPDFPTI JCI85679.f6.jpg F6 FIG fig_title_caption 57380 Comparison of ligated and unligated APLs. 0.9996076 protein_state cleaner0 2023-07-19T20:05:06Z DUMMY: ligated 0.99960905 protein_state cleaner0 2023-07-19T16:17:41Z DUMMY: unligated 0.64326084 chemical cleaner0 2023-07-19T16:29:11Z CHEBI: APLs JCI85679.f6.jpg F6 FIG fig_caption 57422 Superposition of each APL in unligated form and ligated to the 1E6 TCR. All unligated pMHCs are shown as light green illustrations. Peptide sequences are shown underneath each structure aligned with the peptide structure. Black arrows denote the direction of the side chain. Upward arrows indicates solvent exposed, downward arrows indicates anchor position, and no arrow indicates an intermediate position. (A) A2-MVWGPDPLYV (black sticks). A large conformational shift was observed for Tyr8 in the ligated versus unligated states (black circle). (B) A2-YLGGPDFPTI (red sticks). (C) A2-ALWGPDPAAA (blue sticks) reproduced from previous published data. (D) A2-AQWGPDPAAA (green sticks). (E) A2-RQFGPDWIVA (dark blue sticks). (F) A2-RQWGPDPAAV (purple sticks). (G) A2-YQFGPDFPTA (yellow sticks). (H) A2-RQFGPDFPTI (cyan sticks). 0.99966896 experimental_method cleaner0 2023-07-20T08:08:32Z MESH: Superposition 0.9908853 chemical cleaner0 2023-07-19T16:29:17Z CHEBI: APL 0.99965394 protein_state cleaner0 2023-07-19T16:17:41Z DUMMY: unligated 0.99964714 protein_state cleaner0 2023-07-19T20:05:06Z DUMMY: ligated complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9996667 protein_state cleaner0 2023-07-19T16:17:41Z DUMMY: unligated 0.9887504 complex_assembly cleaner0 2023-07-19T16:28:11Z GO: pMHCs 0.9992514 evidence cleaner0 2023-07-19T20:47:54Z DUMMY: structure 0.66176826 evidence cleaner0 2023-07-19T20:48:20Z DUMMY: structure 0.9982674 chemical cleaner0 2023-07-19T16:12:10Z CHEBI: A2-MVWGPDPLYV 0.99989426 residue_name_number cleaner0 2023-07-19T20:04:53Z DUMMY: Tyr8 0.9996716 protein_state cleaner0 2023-07-19T20:05:05Z DUMMY: ligated 0.99966383 protein_state cleaner0 2023-07-19T16:17:41Z DUMMY: unligated 0.99798316 chemical cleaner0 2023-07-19T16:16:11Z CHEBI: A2-YLGGPDFPTI 0.9979634 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.9983786 chemical cleaner0 2023-07-19T18:34:47Z CHEBI: A2-AQWGPDPAAA 0.99870086 chemical cleaner0 2023-07-19T16:12:48Z CHEBI: A2-RQFGPDWIVA 0.9987801 chemical cleaner0 2023-07-19T16:16:24Z CHEBI: A2-RQWGPDPAAV 0.9979759 chemical cleaner0 2023-07-19T18:34:39Z CHEBI: A2-YQFGPDFPTA 0.99866563 chemical cleaner0 2023-07-19T16:12:28Z CHEBI: A2-RQFGPDFPTI JCI85679.f7.jpg F7 FIG fig_title_caption 58250 The 1E6 TCR makes distinct peptide contacts with the MHC surface depending on the peptide cargo. complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9990809 site cleaner0 2023-07-20T07:55:06Z SO: MHC surface JCI85679.f7.jpg F7 FIG fig_caption 58347 Interactions between the 1E6 TCR and the MHC α1 helix residues Arg65, Lys66, and Gln72. Hydrogen bonds are shown as red dotted lines; vdW contacts are shown as black dotted lines. MHCα1 helix are shown in gray illustrations. Boxes show total contacts between the 1E6 TCR and these key residues (green boxes are MHC residues; white boxes are TCR residues). (A) Interaction between the 1E6 TCR (black illustration and sticks) and A2-MVWGPDPLYV (black illustration and sticks). (B) Interaction between the 1E6 TCR (red illustration and sticks) and A2-YLGGPDFPTI (red illustration and sticks). (C) Interaction between the 1E6 TCR (blue illustration and sticks) and A2-ALWGPDPAAA (blue illustration and sticks) reproduced from previous published data. (D) Interaction between the 1E6 TCR (green illustration and sticks) and A2-AQWGPDPAAA (green illustration and sticks). (E) Interaction between the 1E6 TCR (dark blue illustration and sticks) and A2-RQFGPDWIVA (dark blue illustration and sticks). (F) Interaction between the 1E6 TCR (purple illustration and sticks) and A2-RQWGPDPAAV (purple illustration and sticks). (G) Interaction between the 1E6 TCR (yellow illustration and sticks) and A2-YQFGPDFPTA (yellow illustration and sticks). (H) Interaction between the 1E6 TCR (cyan illustration and sticks) and A2-RQFGPDFPTI (cyan illustration and sticks). complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.835831 complex_assembly cleaner0 2023-07-20T07:49:28Z GO: MHC 0.9996875 structure_element cleaner0 2023-07-20T08:11:24Z SO: α1 helix 0.99990416 residue_name_number cleaner0 2023-07-19T18:07:16Z DUMMY: Arg65 0.9999031 residue_name_number cleaner0 2023-07-19T20:05:14Z DUMMY: Lys66 0.999902 residue_name_number cleaner0 2023-07-19T18:10:13Z DUMMY: Gln72 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:18:18Z Hydrogen bonds bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:18:18Z vdW complex_assembly GO: cleaner0 2023-07-20T07:55:49Z MHC structure_element SO: cleaner0 2023-07-20T07:56:02Z α1 helix complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR complex_assembly GO: cleaner0 2023-07-20T07:49:28Z MHC 0.99735093 complex_assembly cleaner0 2023-07-19T20:53:38Z GO: TCR complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9922688 chemical cleaner0 2023-07-19T16:12:10Z CHEBI: A2-MVWGPDPLYV complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9202607 chemical cleaner0 2023-07-19T16:16:11Z CHEBI: A2-YLGGPDFPTI complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.99592036 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9899547 chemical cleaner0 2023-07-19T19:59:25Z CHEBI: A2-AQWGPDPAAA complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9949196 chemical cleaner0 2023-07-19T16:12:48Z CHEBI: A2-RQFGPDWIVA complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.992281 chemical cleaner0 2023-07-19T16:16:24Z CHEBI: A2-RQWGPDPAAV complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.9855128 chemical cleaner0 2023-07-19T19:59:37Z CHEBI: A2-YQFGPDFPTA complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.98272467 chemical cleaner0 2023-07-19T16:12:28Z CHEBI: A2-RQFGPDFPTI JCI85679.f8.jpg F8 FIG fig_title_caption 59707 Thermodynamic analysis of the 1E6 TCR with A2-ALWGPDPAAA and the APLs. 0.9992712 experimental_method cleaner0 2023-07-20T08:08:39Z MESH: Thermodynamic analysis complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.902253 chemical cleaner0 2023-07-19T16:16:50Z CHEBI: A2-ALWGPDPAAA 0.99184835 chemical cleaner0 2023-07-19T16:29:11Z CHEBI: APLs JCI85679.f8.jpg F8 FIG fig_caption 59778 Eight serial dilutions of the 1E6 TCR were injected, in duplicate, over each immobilized APL and A2-ALW at 5°C, 13°C, 18°C, 25°C, 30°C, and 37°C. The equilibrium binding constant (KD) values were calculated using a nonlinear curve fit (y = [P1x]/[P2 + X]), and thermodynamic parameters were calculated according to the Gibbs-Helmholtz equation (ΔG° = ΔH − TΔS°). The binding free energies, ΔG° (ΔG° = RTlnKD), were plotted against temperature (K) using nonlinear regression to fit the 3-parameters van’t Hoff equation (RT ln KD = ΔH° – TΔS° + ΔCp°[T-T0] – TΔCp° ln [T/T0] with T0 = 298 K). (A) 1E6-A2-ALWGPDPAAA; (B) 1E6-A2-AQWGPDPAAA; (C) 1E6-A2-RQFGPDWIVA; (D) 1E6-A2-RQWGPDPAAV, (E) 1E6-A2-YQFGPDFPTA; and (F) 1E6-A2-RQFGPDFPTI. complex_assembly GO: cleaner0 2023-07-19T20:54:52Z 1E6 TCR 0.84157336 chemical cleaner0 2023-07-19T16:29:17Z CHEBI: APL 0.9946752 chemical cleaner0 2023-07-19T20:42:55Z CHEBI: A2-ALW 0.99944896 evidence cleaner0 2023-07-19T20:48:28Z DUMMY: equilibrium binding constant 0.9995074 evidence cleaner0 2023-07-19T16:17:17Z DUMMY: KD 0.97272396 experimental_method cleaner0 2023-07-20T08:08:43Z MESH: nonlinear curve fit experimental_method MESH: cleaner0 2023-07-19T20:00:21Z Gibbs-Helmholtz equation 0.9981754 evidence cleaner0 2023-07-19T20:48:41Z DUMMY: ΔG° 0.99414825 evidence cleaner0 2023-07-19T20:49:03Z DUMMY: ΔH 0.6460132 evidence cleaner0 2023-07-19T20:49:06Z DUMMY: TΔS° 0.9993293 evidence cleaner0 2023-07-19T20:49:08Z DUMMY: binding free energies 0.9992066 evidence cleaner0 2023-07-19T20:48:52Z DUMMY: ΔG° 0.998804 evidence cleaner0 2023-07-19T20:49:00Z DUMMY: ΔG° 0.9961696 experimental_method cleaner0 2023-07-20T08:08:48Z MESH: nonlinear regression experimental_method MESH: cleaner0 2023-07-19T20:02:29Z van’t Hoff equation evidence DUMMY: cleaner0 2023-07-19T20:50:04Z RT ln KD 0.9869153 evidence cleaner0 2023-07-19T20:49:19Z DUMMY: ΔH° evidence DUMMY: cleaner0 2023-07-19T20:02:12Z TΔS° 0.9905539 evidence cleaner0 2023-07-19T20:49:24Z DUMMY: ΔCp° evidence DUMMY: cleaner0 2023-07-19T20:50:20Z TΔCp° 0.99468195 complex_assembly cleaner0 2023-07-19T16:14:43Z GO: 1E6-A2-ALWGPDPAAA 0.9910844 complex_assembly cleaner0 2023-07-19T20:01:23Z GO: 1E6-A2-AQWGPDPAAA 0.99196416 complex_assembly cleaner0 2023-07-19T20:01:31Z GO: 1E6-A2-RQFGPDWIVA 0.99309886 complex_assembly cleaner0 2023-07-19T20:01:39Z GO: 1E6-A2-RQWGPDPAAV 0.992071 complex_assembly cleaner0 2023-07-19T20:01:49Z GO: 1E6-A2-YQFGPDFPTA 0.9890793 complex_assembly cleaner0 2023-07-19T16:19:13Z GO: 1E6-A2-RQFGPDFPTI T2 TABLE table_title_caption 60546 1E6 TCR-pMHC contacts, affinity measurements and thermodynamics complex_assembly GO: cleaner0 2023-07-19T20:03:08Z 1E6 TCR-pMHC 0.9994793 experimental_method cleaner0 2023-07-19T20:03:19Z MESH: affinity measurements experimental_method MESH: cleaner0 2023-07-19T20:03:30Z thermodynamics T1 TABLE table_title_caption 60610 Peptides used in this study