Atypical TRAV1-2 2 T cell receptor recognition of the antigen-presenting molecule MR1

presents vitamin B – related metabolites to mucosal associated invariant T (MAIT) cells, which are characterized, in part, by the TRAV1-2 1 ab T cell receptor (TCR). In addition, a more diverse TRAV1-2 2 MR1-restricted T cell repertoire exists that can possess altered specificity for MR1 antigens. However, the molecular basis of how such TRAV1-2 2 TCRs interact with MR1 – antigen complexes remains unclear. Here, we describe how a TRAV12-2 1 TCR (termed D462-E4) recognizes an MR1 – antigen complex. report the crystal structures of the unliganded D462-E4 TCR and its complex with MR1 presenting the riboflavin-based antigen 5-OP-RU. the TRBV29-1 b -chain of the D462-E4 TCR binds over the F 9 -pocket of MR1, whereby the complementarity-determining region (CDR) 3 b loop and projected into the F 9 -pocket. the CDR3 b loop anchored proximal to the MR1 A 9 -pocket and direct the 5-OP-RU The D462-E4 TCR footprint on MR1 contrasted that of the TRAV1-2 1 topologies MR1-restricted T cell differential modalities greater for specificities.

MR1 presents vitamin B-related metabolites to mucosal associated invariant T (MAIT) cells, which are characterized, in part, by the TRAV1-2 1 ab T cell receptor (TCR). In addition, a more diverse TRAV1-2 2 MR1-restricted T cell repertoire exists that can possess altered specificity for MR1 antigens. However, the molecular basis of how such TRAV1-2 2 TCRs interact with MR1-antigen complexes remains unclear. Here, we describe how a TRAV12-2 1 TCR (termed D462-E4) recognizes an MR1antigen complex. We report the crystal structures of the unliganded D462-E4 TCR and its complex with MR1 presenting the riboflavin-based antigen 5-OP-RU. Here, the TRBV29-1 b-chain of the D462-E4 TCR binds over the F9-pocket of MR1, whereby the complementarity-determining region (CDR) 3b loop surrounded and projected into the F9-pocket. Nevertheless, the CDR3b loop anchored proximal to the MR1 A9-pocket and mediated direct contact with the 5-OP-RU antigen. The D462-E4 TCR footprint on MR1 contrasted that of the TRAV1-2 1 and TRAV36 1 TCRs' docking topologies on MR1. Accordingly, diverse MR1-restricted T cell repertoire reveals differential docking modalities on MR1, thus providing greater scope for differing antigen specificities.
To expand our molecular understanding of how human TRAV1-2 2 TCRs recognize MR1, we determined the crystal structure of unliganded D462-E4 TCR and its complex with the MR1-5-OP-RU adduct. Our data reveal that the TRAV12-2-TRBV29-1 TCR adopts a distinctly different molecular footprint atop MR1 compared with TRAV1-2 1 MAIT TCR-MR1-Ag and MAV36 1 TCR-MR1-Ag ternary complexes. Accordingly, we provide a new understanding of how diverse abTCR usage manifests in differing MR1 docking geometries.
The electron density of 5-OP-RU and residues at the D462-E4 TCR/MR1 molecular interface was unambiguous. Here, the 5-OP-RU ligand in the D462-E4 TCR-MR1-5-OP-RU complex was typically sequestered within the MR1 A9-pocket and formed a Schiff base covalent bond with MR1-Lys-43 (Fig.  4c). The interactions made with the ligand uracil ring, as well as the 29-and 39-OH groups of the ribityl moiety of 5-OP-RU were conserved compared with the TRAV1-2 1 TCR-MR1-5-OP-RU complexes. Nevertheless, the 49-OH and 59-OH groups of the ribityl chain in the D462-E4 TCR-MR1-5-OP-RU structure exhibited two alternate conformations within the pocket, each having 50% occupancy (Fig. 4, c, d, and e). This resulted in greater surface exposure and accessibility of the ribityl moiety, where one conformation was oriented toward the F9 pocket of MR1 and contacted the CDR3b loop. Further, structural modifications within the MR1 antigen binding cleft were observed upon D462-E4 TCR binding compared with the TRAV1-2 1 TCR-MR1 complexes (Fig. 4). Here, part of the MR1 a2-helix (Trp-143 to Asn-155) was slightly displaced (root mean square deviation (rmsd), 0.53 Å) (Fig. 4b), as a result of its interactions with the CDR3b loop compared with the TRAV1-2 1 TCR-MR1 complexes (described below). Collectively, this reflects the adaptability of the MR1 Ag-binding cleft, and its sequestered ligand, upon ligation with the MR1-reactive TCRs.
The CDR1a, CDR2a loops and the Va framework region sat close to the a2-helix and contributed only 6%, 10 and 7% to the complex BSA, respectively. Here, Gln32a of CDR1a formed hydrogen bonds with MR1-Asn155 and Glu160. While Tyr52a from the CDR2a loop extensively interacted with MR1-His148, Tyr152 and Asn155 (Fig. 5c and Table 3). Altogether, the TRAV12-2 TCR a-chain interactions was distinct from that of the TRAV1-2 1 TCR footprint on MR1.
Role of TRBV29-1 b-chain and its CDR3b loop in MR1 recognition The TRBV29-1 b-chain contributed almost two thirds of the interface between D462-E4 TCR and MR1 (61% BSA). The Vb framework region, CDR1b, and CDR2b extended above the a1-helix of MR1 and contributed 7%, 10 and 7% BSA, respectively, to the binding interface. The Vb framework and the CDR1b loop bound MR1 mainly by hydrophobic interactions, whereas Asn-51b from the CDR2b loop H-bonded to MR1-Gln-64 (Table 3 and Fig. 5d).
Surprisingly, the CDR3b loop wedged between the helical jaws of the MR1 cleft, surrounding the F9-pocket, although proximal to the bound antigen in A9-pocket (Fig. 5, e and f). Notably, the CDR3b loop was the principal contributor to the D462-E4 TCR-MR1 interface, providing ;37% of the BSA and thus played a prominent role in the interaction and recognition of D462-E4 TCR by MR1. Moreover, the CDR1b loop played an important role in consolidating the CDR3b loop in a fixed configuration atop the F9-pocket of MR1 by forming H-bonds between Met-32b and Thr-31b from the CDR1b, and the Gly-97b and Asp-99b residues from the CDR3b, respectively.
Here, the 99 Asp-Ser-Leu-Ile-Gly-Asn 104 segment had a dominant role in mediating CDR3b contacts with MR1 (Table 3 and Fig. 5f). The backbone of this peptide folded upon itself to produce a structural "hairpin turn" motif by forming three intramolecular H-bonds (Fig. 5f). This hairpin turn of CDR3b capped the F9-pocket and extensively interacted with various residues of MR1 a1and a2-helices. Interestingly, the CDR3b Asp-99b and Asn-104b residues occupied the space between the A9-and F9-pockets, and their side chains were oriented toward the bound antigen in the A9-pocket. Asp-99b and Ser-100b formed various hydrophobic contacts with the Trp-69, Met-72, Val-75 and Glu-76 of MR1 a1-helix. In addition, the backbone carbonyl of Leu-101b formed H-bonds with the side chains of MR1-Arg-79 and -Trp-143 of a1and a2-helices, respectively. On the other side of the hairpin turn, Ile-102b, Gly-103b, and Asn-104b residues extensively interacted with MR1-Ala-142, -Trp-143, -Asn-146, and -Glu-149 residues from the MR1 a2-helix (Table 3 and Fig. 5f). Accordingly, the CDR3b loop play a prominent role in the interaction and recognition of the D462-E4 TCR by the MR1 molecule.

Divergent recognition of riboflavin derivatives by MR1restricted TCRs
The D462-E4 TRAV1-2 2 TCR presented Asn-97a in an equivalent position to the Tyr-95a of TRAV1-2 1 TCRs. However, the entire CDR3a loop of D462-E4 TCR and its Asn-97a were positioned .6 Å from 5-OP-RU, with no direct or water-  based contacts with ligand observed. Nevertheless, the side chain of MR1-Tyr-152 was H-bonded to the Asn-97a, as well as to the 59-OH of both 5-OP-RU conformations (Fig. 6a). Asp-99b from the CDR3b loop oriented toward the A9pocket of MR1 antigen binding cleft, forming a H-bond (2.9 Å) with the 59-OH of one ribityl conformation (Fig. 6a). The usage of the TRAV1-2 gene in MR1-restricted TCRs facilitates a consistent docking mode atop the MR1. This docking mode maintains the evolutionary conserved Tyr-95a from the CDR3a loop in a conserved location, so that it protrudes deeply into the A9-pocket of the MR1-Ag-binding cleft. This enabled H-bonding between Tyr-95a OH and the 29-OH of the ribityl moiety of the antigen, as well as MR1-Tyr-152 (Fig. 6b)  (2, 3, 28). Indeed, Tyr-95a, Tyr-152 and 5-OP-RU formed an interaction triad that has been shown to play a prominent role in TRAV1-2 1 TCR recognition of MR1-presenting riboflavin derivatives (6). The structure of the MAV36 TCR-MR1-5-OP-RU complex showed that Asn-29a from the CDR1a loop was vital for recognition of 5-OP-RU, by forming a direct H-bond with the 29-OH of the ribityl moiety (Fig. 6c) (30). Notably, the CDR3a Tyr-95a of TRAV1-2 1 TCRs and the CDR1a Asn-29a of MAV36 TCR were closely aligned in a position that enabled their side chains to interact with the 29-OH of 5-OP-RU in a convergent recognition mechanism of the ligand. In contrast, Asp-99b from CDR3b loop of D462-E4 TCR interacted with the terminal 59-OH group of 5-OP-RU. This reveals differential recognition of riboflavin-based metabolites by diverse MR1restricted TCRs.

D462-E4 TCR lock and key recognition of the MR1-5-OP-RU molecule
To investigate the TCR conformational changes upon D462-E4 TCR recognition of MR1-5-OP-RU, we determined the crystal structure of the D462-E4 TCR in its unliganded state ( Table 2 and Fig. 7a) and compared it to the structure of the D462-E4 TCR-MR1-5-OP-RU ternary complex. There was little displacement (rmsd, 0.94 Å) of the variable domain of the a-chain (Va) and all CDRa loops upon binding to MR1 molecule, but with minimal changes to their side chain locations (Fig. 7b). Interestingly, with the exception for CDR3b, no appreciable movement or changes within the CDRb loops were observed after MR1 engagement, suggesting that little conformational adjustments of the CDRb loops were required for recognition of MR1 (Fig. 7, c and d). Indeed, the tip of the CDR3b loop slightly moved (rmsd 0.3 Å) to made favorable contacts with the F9-pocket of MR1. Collectively, the relatively rigid D462-E4 TCR recognized MR1 molecule by a lock and key mechanism.
To examine how other TCRs might bind to MR1, we investigated here the recognition of a TRAV1-2 2 TCR (TRAV12-2-TRBV29-1). This D462-E4 TCR was able to recognize riboflavin-related as well as non-riboflavin-related MR1-bound ligands produced by S. pyogenes microbes (31), which suggested novel D462-E4 TCR docking and recognition strategies for metabolite antigens. Here, we found that this relatively rigid D462-E4 TCR docks centrally onto the MR1 antigen binding cleft but tilts toward and fully caps the F9 portal. Minor changes occurred for the TCR upon complexation with MR1. However, adaptable conformational changes within both the MR1-binding pocket and the 5-OP-RU ligand were observed upon TCR engagement.
Despite the usage of different TRAV genes, both TRAV12-2 of D462-E4 TCR and TRAV1-2 of A-F7 TCRs exhibit similar docking positions atop the A9-pocket of MR1, yet TRAV12-2 contributed much less to the MR1-binding interface (415 Å 2 ) compared with TRAV1-2 (580 Å 2 ). Further, none of the TRAV12-2 loops make contacts with the ligand. Indeed, the b-chain of TRBV29-01, in particular the CDR3b loop, plays a prominent role in D462-E4 recognition of the MR1-5-OP-RU complex. Here, the CDR3b loop extensively interacts with the empty F9-pocket of the MR1 antigen binding cleft. Moreover, Asp-99b from the CDR3b loop of the D462-E4 TCR interacted with the 59-OH group of 5-OP-RU. As such our data demonstrate that the diverse MR1-reactive T cell repertoire exhibits varied docking strategies that enable divergent mechanisms to be used to recognize antigens bound to MR1.

Experimental procedures
Cells and flow cytometry MR1 restricted T cell clones were expanded using anti-CD3 and IL-2 and maintained as previously described (21, 31). Prior to sequencing TCRs, D462-E4 T cell clone was FACS purified after staining with LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies) and an antibody for CD3. TRA and TRB TCR sequencing was performed by immunoSEQ (Adaptive Biotechnologies).
T cell clones were stained with MR1 tetramers (NIH Tetramer Core) for 1 h at room temperature. Cells were then washed with PBS 1 2% FBS buffer and stained with LIVE/ DEAD Fixable Dead Cell Stain Kit (Life Technologies) and surface stained with antibodies specific for CD3, CD4, CD8, and TRAV1-2 for 20 min at 4°C. Samples were fixed with 4% paraformaldehyde for 15 min and washed with a PBS 1 2% FBS buffer, and acquisition was performed using a Fortessa flow cytometer with FACSDiva software (BD Biosciences). All flow cytometry data were analyzed using FlowJo software (Treestar) and Prism (GraphPad).

ELISPOT assays
For the plate-bound tetramer ELISPOT (tetraSPOT) assay, ELISPOT plates were coated with an anti-IFN-g antibody, as described previously (Ref. 31). At the time of coating, MR1 tetramers (NIH Tetramer Core) were added to wells at concentrations between 0 and 5 nM per well. After overnight incubation at 4°C, ELISPOT plates were washed three times with sterile PBS and then blocked with RPMI 1640 1 10% human serum for 1 h. MAIT cell clones (2 3 10 4 ) were added to wells overnight. IFN-g ELISPOTs were enumerated following development as described previously (Ref. 31). For the MR1-blocking tetraSPOT assays, MR1 blocking antibody (clone 26.5) or isotype control (mouse IgG2a) was added at 5 mg/ml in additional wells with the MR1-5-OP-RU tetramer.

Refolding and purification of MR1 and MR1-restricted TCRs
A-F7 (TRAV1-2/TRBV6-1) MAIT TCR and human MR1-b2m-6-FP, Ac-6-FP, and 5-OP-RU were refolded in the cold room overnight in the presence of 0.1 M Tris, pH 8.5, 5 M urea, 2 mM EDTA, 0.4 M L-arginine, 0.5 mM oxidized GSH, and 5 mM reduced GSH as described previously (1,28). Similarly, D462-E4 (TRAV12-2/TRBV29-1) TCR was refolded at 4°C in 1 liter of refold buffer but with three injections of 50 mg of both a TRAV12-2 and b TRBV29-1 chains over 3 days. 5-OP-RU ligand was generated in situ in water from the addition of 5-A-RU and methylglyoxal as previously described (Ref. 2). Next, the refolded MR1-b2m-Ag and TCR proteins were then dialyzed against three changes of buffer containing 10 mM Tris-HCl, pH 8, over 24 h and purified by sequential crude DEAE anion exchange, size exclusion chromatography then HiTrap-Q HP anion exchange chromatography. The purity of the resulting protein was assessed using SDS-PAGE and further quantified by A280 absorbance.

Crystallization, structure determination and refinement
Purified D462-E4 TCR was mixed with MR1-5-OP-RU in a 1:1 molar ratio at a concentration of 8-10 mg/ml and kept on ice for 2 h. Crystals of D462-E4 TCR-MR1-5-OP-RU ternary complex were grown by hanging-drop vapor diffusion method at 20°C, with a reservoir solution containing 18-26% PEG3350, 100 mM Bis-Tris Propane (pH 8.0-8.6) and 200 mM sodium bromide. Similarly, the binary D462-E4 TCR crystals were obtained at concentration of 5 mg/ml with a precipitant consisting of 16-24% PEG3350, 100 mM Bis-Tris Propane (pH 6.0-6.6), and 200 mM sodium fluoride. Both binary and ternary complexes crystals grew within 5-10 days, and then were flashed frozen in liquid nitrogen after quick soaking in reservoir solution with 10-12% glycerol for cryo-protection. X-ray diffraction data were collected at 100 K on the Australian Synchrotron at MX2 beamline (34). Diffraction images were processed using XDS (35) and programs from the CCP4 suite (36) and Phenix package (37). The D462-E4 TCR crystal structure was determined by molecular replacement using PHASER program (38) using an A-F7 TCR as search model (PDB ID: 4L4T). Next, we used the solved binary D462-E4 TCR and MR1 coordinates (PDB ID: 4L4T) as search model to solve the ternary structure of D462-E4 TCR-MR1-5-OP-RU complex. Manual model building was conducted using COOT (39), followed by iterative rounds of refinement using Phenix.refine (37). The Grade Webserver and Phenix tools were used to build and to generate ligand restraints. The models were validated using MolProbity (40) and the final refinement statistics are summarized in Table 2. All molecular graphic representations were generated using PyMOL Molecular Graphics System, Version 1.8, (Schrödinger, LLC, New York, NY). The buried surface area is calculated using AreaIMol program, the contacts generated by the Contact program, both from the CCP4 suite (36).

Data availability
The coordinates of the D462-E4 TCR and D462-E4 TCR-MR1-5-OP-RU crystal structures have been deposited in the Protein Data Bank under accession codes 6XQQ and 6XQP, respectively.