Edited by: Roland Michael Tisch, University of North Carolina at Chapel Hill, United States
Reviewed by: Gustaf Christoffersson, Uppsala University, Sweden; Aaron Michels, University of Colorado, United States
*Correspondence: Jacinto López-Sagaseta,
†These authors have contributed equally to this work and share first authorship
‡Lead Author
This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology
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We recently provided evidence for promiscuous recognition of several different hybrid insulin peptides (HIPs) by the highly diabetogenic, I-Ag7-restricted 4.1-T cell receptor (TCR). To understand the structural determinants of this phenomenon, we solved the structure of an agonistic HIP/I-Ag7 complex, both in isolation as well as bound to the 4.1-TCR. We find that HIP promiscuity of the 4.1-TCR is dictated, on the one hand, by an amino acid sequence pattern that ensures I-Ag7 binding and, on the other hand, by the presence of three acidic residues at positions P5, P7 and P8 that favor an optimal engagement by the 4.1-TCR’s complementary determining regions. Surprisingly, comparison of the TCR-bound and unbound HIP/I-Ag7 structures reveals that 4.1-TCR binding triggers several novel and unique structural motions in both the I-Ag7 molecule and the peptide that are essential for docking. This observation indicates that the type 1 diabetes-associated I-Ag7 molecule is structurally malleable and that this plasticity allows the recognition of multiple peptides by individual TCRs that would otherwise be unable to do so.
Type 1 diabetes (T1D) in nonobese diabetic (NOD) mice results from destruction of pancreatic beta-cells by T-cells targeting a growing list of autoantigens (
Susceptibility to T1D (and other autoimmune diseases) involves a small number of genes with large effects (e.g. Human Leukocyte Antigen (HLA)-coding genes) and a larger number of genes with smaller contributions (
These polymorphisms allow the T1D-associated MHCII molecules to bind to peptides carrying acidic residues at position 9 (P9) (
Transgenic NOD mice expressing the I-Ag7-restricted 4.1-TCR, cloned from islet-associated CD4+ T-cells of diabetic NOD mice, spontaneously develop a dramatically accelerated form of T1D (
The work described herein was undertaken to gain an understanding of the molecular underpinnings underlying the antigenic promiscuity of this TCR. Our crystallographic studies show that the HIP promiscuity of the 4.1-TCR is primarily dictated by (1) the presence of three acidic residues at positions P5, P7 and P8 that help lock a conformationally flexible I-Aβg7 residue away from its position in the TCR-unbound state, which precludes 4.1-TCR docking; and (2) by structural adaptations of several other I-Aαd, I-Aβg7 and peptide residues that promote 4.1-TCR engagement. Collectively, these observations indicate that the pro-diabetogenic I-Ag7 molecule is structurally malleable and suggest that this plasticity might contribute to diabetogenesis by expanding the antigenic repertoire of specific TCRs.
The sequence of the agonist HIP39 antigen lacking its three last amino acids was fused to the I-Aβg7 chain through an Asp>Cys mutation [LQTLALEVEDDPC] to enable covalent linkage through a disulfide bridge with a concomitant Ile74Cys replacement in I-Aαd.
HIP39/I-Ag7 monomers were expressed and produced in lentiviral-transduced CHO-S cells as knob-into-hole (KIH) Fc fusions (
Production of the 4.1-TCR and HIP39/I-Ag7 complexes. Top, lentiviral constructs encoding the HIP39/I-Ag7. The HIP39 peptide sequence is tethered to the I-Aβg7 chain
Structure of I-Ag7 with a bound agonistic hybrid insulin peptide (HIP39).
The HIP39/I-Ag7 complex crystallized in space group C2221 with a single pMHCII molecule per asymmetric unit (
Diffraction data collection and refinement statistics.
4.1-TCR : HIP39/I-Ag7 | HIP39/I-Ag7 | |
---|---|---|
|
49.74 - 2.65 (2.745 - 2.65) | 54.41 - 1.82 (1.885 - 1.82) |
|
C121 | C2221 |
|
213.615 133.457 102.56 90 103.689 90 | 91.996 108.819 98.055 90 90 90 |
|
160776 (15963) | 243215 (24982) |
|
81034 (8068) | 44329 (4392) |
|
2.0 (2.0) | 5.5 (5.7) |
|
99.83 (99.78) | 99.86 (99.98) |
|
8.17 (1.15) | 13.17 (1.66) |
|
58.56 | 29.68 |
|
0.0698 (0.742) | 0.07441 (1.009) |
|
0.0987 (1.05) | 0.0825 (1.111) |
|
0.0698 (0.742) | 0.03513 (0.4603) |
|
0.994 (0.435) | 0.998 (0.647) |
|
81008 (8068) | 44326 (4392) |
|
4063 (414) | 2213 (215) |
|
0.2212 (0.3165) | 0.1855 (0.2663) |
|
0.2612 (0.3597) | 0.2178 (0.3185) |
|
0.916 (0.600) | 0.963 (0.828) |
|
0.886 (0.485) | 0.951 (0.737) |
|
0.002 | 0.006 |
|
0.53 | 0.80 |
|
97.3 | 98.92 |
|
2.7 | 1.08 |
|
0.06 | 0.00 |
|
61.91 | 33.68 |
Statistics for the highest-resolution shell are shown in parentheses.
The I-Ag7-restricted 4.1-TCR was originally isolated from autoreactive CD4+ T lymphocytes infiltrating the pancreatic islets of NOD mice. The α and β chains are encoded by TRAV5D-4*04/TRAJ40*01 and TRBV16*01/TRBJ2-4*01 VDJ rearrangements, respectively. For crystallization studies, the variable regions of the α and β chains were fused with the corresponding human TCR constant regions mutated to encode a pair of heterodimerizing cysteines. TCRα and β chains were folded jointly from inclusion bodies, purified and incubated with recombinant HIP39/I-Ag7 complexes prior to crystallization trials. Plate-like crystals were used to collect a 2.65 Å full native dataset that enabled solution of the ternary complex structure. Crystals belonged to the C121 space group and two equivalent 4.1-TCR : HIP39/I-Ag7 complexes (RMSD 0.216) were obtained in the asymmetric unit (
Structure of the 4.1-TCR : HIP39/I-Ag7 complex.
The TCR-pMHCII interface covers an average buried surface area (BSA) of 1172 A2, a value somewhat lower than that reported for most other TCR-pMHCII interfaces.
Importantly, most of the residues in I-Aαd and I-Aβg7 that make contacts with the TCR are conserved across murine MHCII molecules. The I-Aαd chain contributes to TCR binding with multiple residues and contacts of various types (
Intermolecular contacts between the 4.1-TCR and HIP39/I-Ag7.
The I-Aβg7 residues that participate in interactions with the 4.1-TCR (
We have recently shown that the 4.1-TCR can recognize 6 additional HIPs, in addition to HIP39 (HIPs 15, 18, 30, 30 Q10E, 32 and 32 Q9E), conforming to the following nonamer motif: TLALE(V/A/G)E(D/E)(D/E/P/Q). In all these HIPs, the left arm is donated by the Insulin C-peptide (InsC), truncated at different carboxyterminal residues, whereas the right arm corresponds to different naturally occurring proteolytic products of either Chromogranin A (ChgA) or InsC57-63. HIP30Q10E and HIP32Q9E correspond to the deamidated forms of HIPs 30 and 32, respectively. All these HIPs promoted IFNγ secretion by activated 4.1-CD4+ T-cells, albeit with different functional avidities. Experiments using pMHCII tetramers indicated that these 7 HIPs could be further subclassified into three different subsets: one with high functional avidity and strong tetramer binding avidity (HIP39 and HIP32Q9E); a second subset having a slightly lower functional avidity but only marginal tetramer binding avidity (HIP32 and HIP30Q10E); and a third subset displaying low functional and physical binding avidities (HIP30, HIP15 and HIP18). A fundamental difference between groups 1 and 2 lies in the replacement of a negatively charge amino acid (D or E) at P9, involved in I-Ag7 binding, with P or Q. Unlike tetramer binding, which requires the display of the peptide on the same register on its four monomers, the IFNγ secretion assay can withstand register multiplicity, particularly at high concentration of soluble peptide (
Collectively, these data suggested that positions E5, E7 and D8 are key 4.1-TCR contact residues. Close analysis of the ternary structure described herein demonstrates that this is indeed the case. The first noticeable feature is that E5 and D8 are solvent exposed and, therefore, easily accessible for TCR recognition (
Molecular recognition of the agonistic I-Ag7-presented HIP39 antigen by the diabetogenic 4.1-TCR.
Collectively, these observations indicate that the presence of concurrent acidic residues at positions 5, 7 and 8 in peptides anchored onto I-Ag7 enables autorecognition by the 4.1-CD4+ T-cell.
Availability of the TCR-bound and free HIP39/I-Ag7 structures afforded a unique opportunity to investigate whether promiscuous recognition of HIP/I-Ag7 complexes by the 4.1-TCR involved structural adaptations of the pMHCII and/or the TCR. At first sight, no remarkable differences were noticed in the unbound HIP39/I-Ag7 complex as compared to its 4.1-TCR-bound counterpart, consistent with an overall RMSD of 0.383 for the HIP39/I-Ag7 complex. However, comparison of amino acid side chain positioning in the two structures revealed that 4.1-TCR engagement involves subtle, yet critical motions of certain I-Aβg7 residues’ side chains without which TCR engagement would not be possible.
The most obvious conformational change involves I-Aβg7’s Arg68, which switches the positioning of its side chain from an initial arrangement away from I-Ag7’s peptide-binding groove, to a locked and HIP39-oriented conformation in the presence of the 4.1-TCR (
Structural plasticity of both I-Ag7 and peptide contribute to 4.1-TCR docking.
This positioning of Arg68’s side chain away from the peptide-binding groove is also seen in other pI-Ag7 crystal structures in the absence of a TCR (
Other relevant structural motions are observed in I-Aαd residues Glu57, Gln59 and Glu72 (
We also observed mild, yet pivotal structural transitions in the HIP39 peptide residues that occur in synch with the Arg68 switch discussed above (
To gain further insights into the driving forces behind HIP recognition, we explored the impact of 4.1-TCR binding in the energy signature of the pMHCII complex. To accomplish this purpose, we examined the energy values in HIP39/I-Ag7 in a per-residue basis both on the absence or presence of the 4.1-TCR using a prediction algorithm (pyDockEneRes, see methods) focused on electrostatics, Van der Waals and third component related with desolvation energy. By following this approach, a severe impact was readily noticeable in the HIP antigen. This effect was particularly relevant for residue Glu5, whose energy gain reached -12.5 kcal mol-1 in the presence of the 4.1-TCR (
In sum, our data expose a previously unrecognized role for structural malleability of certain pMHCII complexes in TCR binding, long thought to be largely a property of the TCR CDR loops (
Compelling experimental evidence have suggested that T1D initiation/progression involves the initial recognition of insulin/HIP epitopes followed by the recruitment of autoreactive T-cells targeting many other beta cell autoantigens (
We have shown that transgenic NOD and NOD.
Although the overall morphology of the I-Ag7-MHC molecule is preserved in both the free and TCR-bound structures, the side chains of several residues in I-Ag7 dramatically changed their orientation upon TCR binding. Particularly striking were the rearrangements involving Gln59 in I-Aαd and Arg68 in I-Aβg7. These rearrangements not only eliminated intermolecular clashes precluding TCR binding but also enabled the formation of new interactions that contributed to tighter TCR engagement. Of note, the Arg68 rearrangement was accompanied by a subtle yet key movement of the HIP’s E5 residue that resulted in proper positioning of the 4.1-TCR’s CDR3β on the HIP39/I-Ag7 complex. Additional key, albeit less pronounced shifts were noted for I-Aαd’s Glu57 and I-Aβg7’s Glu72, which also enabled stronger interactions with the 4.1-TCR through novel
An alternative interpretation of the data is that the monomeric pMHCII preparations used for crystallization of ternary pMHCII-TCR complexes correspond to heterogeneous populations of structurally diverse pMHCII monomers, each with alternative conformations in the side chains of specific amino acids. Under this hypothetical scenario, only a fraction of conformationally-compatible and energetically favorable pMHCII complexes would license 4.1-TCR engagement and crystallization. However, in-depth analyses of other pI-Ag7-TCR crystal structures, such as those corresponding to the Insulin B:9-23-specific I.29/8F10 and HEL-specific 21.20 TCRs, respectively, strongly argue against this alternative interpretation of the data. As noted further above, the positioning of Arg68’s side chain away from the peptide-binding groove can also be seen in other pI-Ag7 crystal structures in the absence of a TCR, or in TCR-bound pI-Ag7 structures in which the peptide’s P5 and P7 positions are occupied by neutral residues, as is the case for the I.29 and 8F10 TCR ligands. In contrast, the Arg68’s side chain is found pointing towards the peptide-binding groove in the structure of a HEL peptide/I-Ag7 complex bound to its cognate 21.30 TCR. A key difference between these structures is that P5 and P7 in the HEL peptide are occupied by Glu and Tyr, respectively, which lock Arg68 through a set of polar interactions. Furthermore, limited and isolated displacement of the Arg68 side chain can also be seen in the crystal structures of a pI-Ab or a pI-Au molecules upon engagement of their respective cognate TCRs, in both cases in a peptide-independent manner (
Recent structures of human TCRs bound to a HIP/HLA-DQ8 pMHCII complex (
In addition to the structural analysis, we assessed the energetic yield of the 4.1-TCR : HIP39/I-Ag7 interaction in a per-residue basis. The most outstanding finding were the net energy gain values observed upon binding of the 4.1-TCR, for the glutamic acids in P5 and P7 of the HIP antigen. At the structural level, we observed that these residues acquire conformations whereby the side chains are solvent accesible,
We also noticed that the conformational changes observed for certain side chains in I-Aαd and I-Aβg7 do not necessarily represent energetically favorable transitions. For instance, the conformation of the side chain of Glu57 in I-Aαd in the presence of the 4.1-TCR shows a penalty of 1.4 kcal mol-1 with respect to that of the TCR-unbound structure. Gln59 in I-Aαd presents a weak energy gain in the presence of the TCR [-0.08 kcal mol-1], while the conformation of Glu72 side chain in I-Aβg7 has a more evident energy benefit [-1.2 kcal mol-1], likely due to the possibility of establishing close salt bridges with CDR3α Arg96.
Collectively, our findings add another level of complexity to previous observations of conformational changes in TCRs, MHC molecules and MHCII-bound peptides. The TCR CDR loops are known to undergo structural rearrangements to recognize peptides and lipids presented by classical (
The 4.1-TCR variable domains were fused
HIP39/I-Ag7 monomers were expressed and produced in CHO-S cells (Invitrogen). Briefly, CHO-S cells were transduced sequentially with lentiviral particles encoding a HIP39 peptide-tethered, knob-into-hole (KIH)-based pMHCII heterodimer carrying a 3C cleavage site immediately upstream of each of the two KIH halves (HIP39-linker GS-I-Aβg7–HRV3C-Fc(Hole)-6xHisTag-IRES-GFP and I-Aαd–HRV3C-Fc(Knob)-6xHisTag-IRES-CFP. Sorted CFPhi/GFPhi CHO-S cells were cultured as described (
Crystallization was accomplished by manual screening of over 700 crystallization conditions for each protein sample in 96-well plates using the sitting drop method and 20 °C incubation. Crystals of HIP39/I-Ag7 appeared in 24-48 hours and had needle or rod-shaped morphology. Large optimized three-dimensional crystals were obtained in 0.17 M ammonium sulfate and 25% w/v PEG 4000. Square-shaped thin crystals of 4.1-TCR : HIP39/I-Ag7 appeared after 2-4 weeks at 20 °C and were optimized in 0.1 M MgCl2, 0.1 M Na HEPES pH 7.0, 15% w/v PEG 4000. Crystals were harvested, soaked in their crystallization medium supplemented with 20% glycerol (HIP39/I-Ag7) or 20% ethylenglycol (4.1-TCR : HIP39/I-Ag7) and transferred to liquid nitrogen prior to diffraction analyses.
Crystals were mounted in the diffractometer at the Xaloc beamline of the Alba Synchrothron (Cerdanyola del Vallès, Barcelona, Spain). Diffraction datasets were collected for HIP39/I-Ag7 and 4.1-TCR : HIP39/I-Ag7 crystals. X-ray diffraction data of HIP39/I-Ag7 crystals were processed with autoPROC (
The atomic coordinates of the HIP39/I-Ag7 and 4.1-TCR : HIP39/I-Ag7 complex structures were used to calculate energy values in a per-residue basis with pyDockEneRes (
Atomic coordinates and structure factors of the HIP39/I-Ag7 and 4.1-TCR:HIP39/I-Ag7 complexes have been deposited in the Protein Data Bank with accession codes 7QHP and 7Z50, respectively.
Conceptualization: JL-S and PSa. Methodology: JL-S, PSe and PSa. Experimental research: EE, JL-S, DP, PSe. Manuscript writing: JL-S and PSa. All authors contributed to the article and approved the submitted version.
JL-S is a Ramón y Cajal Investigator supported by the Ministerio de Ciencia e Investigación of Spain (RYC‐2017‐21683). DP was supported by a pre-doctoral studentship from FPU (MINECO). This work was supported by the Ministerio de Ciencia e Investigación of Spain (RTI2018-093964-B-I00), Generalitat de Catalunya (SGR and CERCA Programmes), the ISCIII and FEDER (PIE14/00027, PI15/0797), and the Canadian Institutes of Health Research (CIHR-136866). PSe was supported by the Ramon y Cajal program and by a JDRF Career Development Award.
We thank the staff of XALOC beamline at ALBA Synchrotron for their assistance with X-ray diffraction data collection. We thank M.G. Dichiara Rodríguez, A. Ochoa Echeverria, C. Fandos, M. Ortega, S. Thiessen and F. Liu for technical support.
PSa is scientific founder of Parvus Therapeutics and has a financial interest in the company.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The Supplementary Material for this article can be found online at:
Crystal samples and composition of the asymmetric unit in the HIP39/I-Ag7 and 4.1-TCR:HIP39/I-Ag7 ternary complexes.
Multiple contacts mediated by I-Aβg7 Thr75 with the 4.1-TCR. Thr75 engages with all CDR loops in the 4.1-TCRα chain. Residues in the CDRs making contacts are shown as sticks. VDW contacts are displayed as blue dash lines, while the H-bond with Ser53 can be seen in darkgrey color. All contacts are < 4.0 Å.
Additional contacts mediated by water molecules between the 4.1-TCR and the HIP39/I-Ag7 complex.
Structural plasticity of I-Aβg7 Arg68.
Energy balance of the HIP39/I-Aβg7 complex upon recognition by the 4.1-TCR. Electrostatics, Van der Waals forces and desolvation terms were computed for both the HIP antigen and the MHCII in a per-residue basis with pyDockEneRes (see methods). The net energy gain for the HIP39/I-Aβg7 complex was assessed with the difference of the calculated values in the presence and absence of the TCR. The graphic shows the net energy gain values in a per-residue manner for the HIP antigen (residues -1 to 11), I-Aαd and I-Aβg7.
I-Aαd chain with HIP39 interactions found in 7QHP. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aβg7 chain with HIP39 interactions found in 7QHP. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aαd (chain A) with HIP39 (chain T) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å, salt bridges ≤ 4.5 Å, Van der Waals ≤ 4.0 Å.
I-Aαd (chain C) with HIP39 (chain W) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aβg7 (chain B) with HIP39 (chain T) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aβg7 (chain D) with HIP39 (chain W) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
4.1-TCR α (chain H) with HIP39 (chain T) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
4.1-TCR α (chain G) with HIP39 (chain W) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
4.1-TCR β (chain E) with HIP39 (chain T) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
4.1-TCR β (chain F) with HIP39 (chain W) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aαd (chain A) with 4.1-TCR α (chain H) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aαd (chain C) with 4.1-TCR α (chain G) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aαd (chain A) with 4.1-TCR β (chain E) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aαd (chain C) with 4.1-TCR β (chain F) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aβg7 (chain B) with 4.1-TCR α (chain H) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aβg7 (chain D) with 4.1-TCR α (chain G) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aβg7 (chain B) with 4.1-TCR β (chain E) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å
I-Aβg7 (chain D) with 4.1-TCR β (chain F) interactions found in 7Z50. All intermolecular contacts are assigned according to the following distance cutoffs: hydrogen bonds ≤ 3.4 Å