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Antigen Bound to MR1MAIT Recognition of a Stimulatory Bacterial

AdamsMcFedries, Mark Cushman, Alan Saghatelian and Erin J. Jacinto López-Sagaseta, Charles L. Dulberger, Amanda

ol.1301958http://www.jimmunol.org/content/early/2013/10/08/jimmun

published online 9 October 2013J Immunol 

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The Journal of Immunology

MAIT Recognition of a Stimulatory Bacterial Antigen Boundto MR1

Jacinto Lopez-Sagaseta,* Charles L. Dulberger,* Amanda McFedries,† Mark Cushman,‡

Alan Saghatelian,† and Erin J. Adams*,x

MR1-restricted mucosal-associated invariant T (MAIT) cells represent a subpopulation of ab T cells with innate-like properties

and limited TCR diversity. MAIT cells are of interest because of their reactivity against bacterial and yeast species, suggesting that

they play a role in defense against pathogenic microbes. Despite the advances in understanding MAIT cell biology, the molecular

and structural basis behind their ability to detect MR1–Ag complexes is unclear. In this study, we present our structural and

biochemical characterization of MAIT TCR engagement of MR1 presenting an Escherichia coli–derived stimulatory ligand, rRL-

6-CH2OH, previously found in Salmonella typhimurium. We show a clear enhancement of MAIT TCR binding to MR1 due to the

presentation of this ligand. Our structure of a MAIT TCR/MR1/rRL-6-CH2OH complex shows an evolutionarily conserved

binding orientation, with a clear role for both the CDR3a and CDR3b loops in recognizing the rRL-6-CH2OH stimulatory

ligand. We also present two additional xenoreactive MAIT TCR/MR1 complexes that recapitulate the docking orientation

documented previously, despite having variation in the CDR2b and CDR3b loop sequences. Our data support a model by which

MAIT TCRs engage MR1 in a conserved fashion, with their binding affinities modulated by the nature of the MR1-presented Ag

or diversity introduced by alternate Vb usage or CDR3b sequences. The Journal of Immunology, 2013, 191: 000–000.

Mucosal-associated invariant T (MAIT) cells are anevolutionarily conserved subpopulation of ab T cellsin mammals that are characterized by the expression

of a semi-invariant ab TCR and restriction to the MHC-likeprotein MR1 (1–3). MAIT cells exhibit an innate-like phenotypeand are found predominantly in mucosal tissues, such the gutlamina propria and the lung. MAIT cells are also found at highfrequency in the liver, and in blood they can represent up to 10%of the CD42 ab T cell population (4, 5). MAIT cell developmentis dependent on MR1 expression, B cells, and an established hostcommensal flora (2, 6).Highly conserved across mammalian evolution, MR1 transcripts

and protein are detected in most tissues, yet detection on the cellsurface is low or undetectable under basal conditions (7, 8). Therecent structural elucidation of MR1 (9, 10) revealed an overallbackbone structure similar to peptide-presenting, classical MHCmolecules but with a putative Ag-binding cavity of smallerdimensions due to the presence of large aromatic and basic resi-

dues lining the cavity. The aromatic architecture of this cavity iscomplementary to the small, ring structure containing ligandsidentified recently (10) that are derivatives of two B vitamin met-abolic pathways: folic acid (vitamin B9) and riboflavin (vitaminB2). 6-Formylpterin (6-FP) was identified as an MR1-bound ligand,potentially covalently attached via Schiff base to lysine 43 foundin the ligand-binding cavity of MR1. 6-FP was nonstimulatory toMAIT cells. In contrast, 6,7-dimethyl-8-ribityllumazine (DMRL),the direct precursor to riboflavin, and two DMRL variants wereidentified as MR1 ligands sufficient to stimulate MAIT cells.To engage an MR1–Ag complex, human MAIT cells use an ab

TCR composed of a mostly invariant a-chain (Va7.2/Ja33) thatassociates with a limited array of b-chains: Vb2, Vb13, andVb22, among others (11). Diversity in the variable domain of theb-chain arises from Vb-Db-Jb gene segment rearrangement thatgenerates highly variable CDR3b loops, conferring MAIT TCRsa certain level of diversity despite the Vb restriction. MAIT cellscan be activated upon infection with diverse bacterial or yeastspecies, including Escherichia coli, Staphylococcus aureus,Mycobacterium tuberculosis, Salmonella typhimurium, Candidaalbicans, and Saccharomyces cerevisiae, but not viruses (5, 12).Human MAIT cell autoreactivity was also described in MR1-transfected cells in a microbial-independent manner (7, 13, 14),suggesting that endogenous ligands can contribute to MAIT cellreactivity.Taking advantage of a ligand-independent, cross-species reac-

tivity between human MAIT cells and bovine MR1, our grouprecently reported the first crystal structure of a complex betweena MAIT TCR and MR1, providing a molecular model for howMAIT cells engage their MR1 ligand (9). This model was con-sistent with mutagenesis of the MAIT TCR/MR1 interaction fromother groups (14, 15), and superimposes almost identically witha recently reported complex structure of a human MAIT TCR withhuman MR1 (16), supporting it as a bona fide model for MAITTCR engagement of MR1. In this complex, the MAIT TCR boundin a diagonal fashion, reminiscent of classical ab TCR recognitionof MHC–peptide complexes (17) and type II NK T cell recogni-

*Department of Biochemistry and Molecular Biology, University of Chicago, Chicago,IL 60637; †Department of Chemistry and Chemical Biology, Harvard University,Cambridge, MA 02138; ‡Department of Medicinal Chemistry and Molecular Pharma-cology, College of Pharmacy, Purdue University, West Lafayette, IN 47907; andxCommittee on Immunology, University of Chicago, Chicago, IL 60637

Received for publication July 24, 2013. Accepted for publication September 5, 2013.

This work was supported by National Institutes of Health Grant R01AI073922 (toE.J.A.).

Coordinates and structure factors have been submitted to the Protein Data Bank(http://www.rcsb.org/) under accession numbers 4LCC (F7 MAIT TCR/hbMR1),4L8S (G2 MAIT TCR/bMR1), and 4L9L (AE6 MAIT TCR/bMR1).

Address correspondence and reprint requests to Dr. Erin J. Adams, Department ofBiochemistry and Molecular Biology, University of Chicago, 929 East 57th Street,GCIS W236, Chicago, IL 60637. E-mail address: ejadams@uchicago.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: DMRL, 6,7-dimethyl-8-ribityllumazine; 6-FP,6-formylpterin; hbMR1, humanized version of bovine MR1; MAIT, mucosal-associ-ated invariant T; MS, mass spectrometry; rRL-6-CH2OH, reduced 6-hydroxymethyl-8-(1-D-ribityl)lumazine; VDW, van der Waals.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301958

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tion of CD1d (18, 19). This docking orientation positioned theCDR3 loops of both the a2 and b-chains close to the opening ofthe ligand-binding cavity. Modeling of the stimulatory ligandsidentified by Kjer-Nielsen et al. (10) into this complex suggestedan important role for Tyr95 of the CDR3a loop, because thisresidue was positioned directly over the opening of this cavity, inhydrogen-bonding proximity to the ribityl chain of the stimulatorycompounds DMRL and reduced 6-hydroxymethyl-8-(1-D-ribityl)lumazine (rRL-6-CH2OH). This complex also identified the threeresidues on MR1 mediating human/cow cross-reactivity.In this study, we present our results derived from an engineered,

humanized version of bovine MR1 (hbMR1) that was expressedrecombinantly in insect cells exposed to E. coli culture supernatantas a source of MAIT cell–reactive ligands. We demonstrate anenhancement of MAIT TCR binding to E. coli–loaded hbMR1over unloaded hbMR1 and show using mass spectrometry (MS)that the bound MR1 ligand is rRL-6-CH2OH. Furthermore, ourcomplex of the F7 MAIT TCR with this rRL-6-CH2OH–loadedMR1 provides critical information about how the MAIT TCRengages MR1 ligands, confirming a pivotal role for Tyr95 of theCDR3a loop and establishing a role for the CDR3b loop in ligandbinding. This complex, like our previously reported xenoreactivecomplex, is strikingly similar to the recently reported human MAIT/MR1 complex with a highly similar stimulatory compound (16),reinforcing the evolutionary conservation of this interaction. Last, wepresent structural evidence that variation in Vb usage and diversityin the sequence of the CDR3b loops does not affect TCR docking,but it can modulate the affinity of binding to MR1, with and withoutstimulatory ligand, through variable contacts with MR1 and ligand.

Materials and MethodsRecombinant protein expression and purification

hbMR1 was cloned, expressed, and purified as previously described (9).Briefly, the triple mutant (hbMR1) was generated through overlappingPCR and cloned into the pAcGP67A vector (BD Biosciences) for subse-quent expression via baculovirus in Hi5 cells. The recombinant protein waspurified with Nickel NTA Agarose (QIAGEN), anion exchange, and size-exclusion chromatography. For preparation of E. coli supernatant, BL21cells were cultured overnight and then centrifuged at 6000 rpm for 30 min.The supernatant was then passed through a 10-kDa cut-off membrane, andthe flow-through was collected. Variable amounts (10–100 ml) of this flow-through were added every 24 h to the Hi5s cells postinfection with therecombinant baculovirus for hbMR1. The expression and purification ofthe MAIT TCR clones F7, G2, and AE6 were carried out in a similar wayto previously described procedures for both Hi5 and E. coli–expressionsystems (9, 20, 21).

Binding studies of MR1 and MAIT–TCR interactions

For binding analysis between hbMR1 and the different human MAIT TCRclones, hbMR1 expressed in the presence or absence of the E. coli su-pernatant was captured to 4 hm units on a Ni-NTA (NTA) Biosensorin a Blitz System Package (Fortebio), as described previously (9). Thebinding affinities for the F7, G2, and AE6 MAIT TCR clones were eachtested by running increasing concentrations of the TCRs over the immo-bilized hbMR1 protein in 10 mM HEPES (pH 7.4), 150 mM NaCl. IgG FCwas captured to a similar level and used to subtract nonspecific bindingsignals. Subtracted responses were then used for calculating KD withGraphPad Prism by plotting the binding values at equilibrium against theTCR concentrations.

MS analysis of hbMR1

For hbMR1, comparison of hbMR1 protein that was exposed to E. colisupernatant and an untreated control hbMR1 sample were analyzed on aBruker maXis impact QTOF LC/MS operating in the negative ion mode.Five microliters each of 25 mM hbMR1 was injected onto a Luna NH24.6 3 50-mm, 5-mm column in 20 mM ammonium acetate (pH 9) bufferand eluted with an aqueous gradient, as described above. The retention timeof rRL-6-CH2OH was determined by extracted ion chromatograms usingthe m/z values. Product ions were obtained from target fragmentation atcollision-induced voltages of 20eV.

Ternary complex formation and crystallization

For crystallization purposes, F7, G2, and AE6 recombinant E. coli–derivedheterodimeric MAIT TCRs were refolded and purified (9), stoichiometri-cally mixed with hbMR1 or bovine MR1, and concentrated to 5–10 mg/ml.Crystals of bovine MR1/F7 MAIT TCR complex were used for micro-seeding fresh sitting drops containing hbMR1/F7 MAIT TCR, bovineMR1/G2 MAIT TCR, and bovine MR1/AE6 MAIT TCR and mother li-quor consisting of 100 mM HEPES and various concentrations of am-monium sulfate (0.9–2.0 M). Crystals for all of the complexes appeared attimes ranging from 2 to 12 wk.

Crystallographic data collection, structure determination, andrefinement

Prior to data collection, crystals were soaked in mother liquor containing20% glycerol and then cryo-cooled. All data sets were collected ona MAR300 CCD at beamline 23 ID-D and 23 ID-B at the Advanced PhotonSource at Argonne National Laboratory (Lemont, IL) and processed withHKL2000 (22). All datasets were corrected for anisotropic diffraction withthe Phenix software suite (23). The structures of the ternary complexeswere solved using the published complex structure of bovine MR1/F7MAIT TCR (PDB 4IIQ) as a search model and removing the TCR CDRloops and all nonprotein coordinates prior to molecular replacement withPhaser (24). The derived solution was refined with the Phenix.refinepackage (23), including Translation/Libration/Screw vibrational motions(25) at the latest stages of refinements, and combined with manual buildingin Coot (26) in between the refinement steps. When necessary, PRODRG(27) was used for the generation of ligand libraries and coordinates thatwere included in the refinement and building process. A random 5% ofreflections were taken out of each data set for statistical validation pur-poses (Rfree) throughout the entire refinement process.

Structure analysis

Intermolecular contacts and distances were calculated using the programContacts from the CCP4 software package (28), interface surface areaswere calculated using the PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html), and all structural figures were generated using theprogram Pymol (Schrodinger). Coordinates and structure factors have beensubmitted to the Protein Data Bank (http://www.ncbi.nlm.nih.gov/pubmed/10592235) under accession codes 4LCC (F7 MAIT TCR/hbMR1), 4L8S(G2 MAIT TCR/bMR1), and 4L9L (AE6 MAIT TCR/bMR1).

ResultsMAIT TCRs show increased affinity for hbMR1 expressed inthe presence of E. coli supernatant

Our initial attempts to recombinantly express human versions ofMR1 in insect cells failed despite extensive attempts at engineer-ing different constructs. Alternatively, we generated a humanizedversion of bovine MR1 (9), hbMR1, by mutating the three TCRcontact positions that differ between human and bovine MR1(Ala72Met in MR1 a1 helix and Arg147Gln and Gln151Leu in a2helix), which we showed modulated MAIT cell xenoreactivity (9).This construct was expressed well and eluted as a monodispersedpeak under size-exclusion chromatography. We used the constructin this study to characterize, structurally and biophysically, therecognition of a MAIT cell–reactive ligand bound to hbMR1 byhuman MAIT TCRs.Several studies demonstrated MAIT cell reactivity against MR1-

transfected APCs infected with different strains of bacteria and/oryeast (5, 10, 12, 14). One of the strains able to trigger MAIT cellresponse and activation is E. coli. We sought to determine whethera stimulatory ligand could be loaded by expressing, in our insect-expression system, our recombinant hbMR1 in the presence ofE. coli supernatant. Protein derived from this method had a no-ticeable yellow hue, consistent with the presence of a flavonoidsubstance. Using bio-layer interferometry, we compared the bind-ing kinetics of three MAIT TCRs to the E. coli–loaded hbMR1compared with that of hbMR1 expressed in the absence of E. colisupernatant (Fig. 1). The binding studies revealed a 20-fold in-crease in the binding affinity by the F7 MAIT TCR, a .100-fold

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increase by the G2 MAIT TCR, and a low micromolar affinityinteraction for the AE6 MAIT TCR clone, whose interaction withthe untreated hbMR1 was not measureable (Fig. 1). These affin-ities (∼4.5 and 4.9 mM) measured for the MAIT TCR interactionswith hbMR1 loaded with a ligand derived from E. coli supernatantare similar to those measured for MAIT TCRs with human MR1loaded with synthetic rRL-6-CH2OH (1.65 mM) (16). Differencesin MR1-production strategies and ligand loading between thesetwo studies might have resulted in slight differences in affinity.These results suggest that hbMR1 is loaded with a ligand or ligandsof bacterial origin that play a direct role in the enhancement ofrecognition by the MAIT TCR.

The E. coli–derived ligand bound by hbMR1 is rRL-6-CH2OH

These results prompted us to useMS to analyze the content of hbMR1protein in samples that were exposed or not to E. coli. Normal-phasequadrupole mass analyzer time-of-flight mass analyzer analysisclearly identified a compound with a retention time ∼ 7 min presentonly in the E. coli–treated sample (Fig. 2A). MS analysis of this

compound revealed it to have an m/z ratio of 329.1095, and itsfragmentation yielded a pattern nearly identical to that found forrRL-6-CH2OH (Fig. 2B), a MAIT cell stimulatory ligand charac-terized from S. typhimurium supernatant (10). This compound isgenerated as a by-product of the riboflavin synthesis pathway and itsstructure consists of a lumazine core and a ribityl chain (Fig. 2A,inset). rRL-6-CH2OH was shown to trigger MAIT cell activation inan MR1-dependent manner, and it displayed the strongest potency inactivating MAIT cells among three structurally related compounds(10). Thus, the increased MAIT TCR affinity for the E. coli–treatedhbMR1 sample correlates with the presence of this ribityl moiety,which our model suggested (9) participates directly in MAIT TCRrecognition of MR1-presented Ag.

The crystal structure of F7 MAIT TCR in complex with hbMR1and rRL-6-CH2OH demonstrates a conserved bindingorientation

To determine the structural basis for this enhanced recognition ofrRL-6-CH2OH–loaded hbMR1 by the MAIT TCR, we used our

FIGURE 1. Expression of hbMR1

in the presence of E. coli superna-

tant enhances the recognition by

three human MAIT TCRs. Associa-

tion and dissociation binding curves

measured by biolayer interferometry

of three human MAIT TCRs with

hbMR1 expressed in the presence

(+) or absence (-) of E. coli superna-

tant (upper panels). Binding curves

for the highest concentrations of

MAIT TCR tested (60 mM) are shown

for comparison. Nonlinear regression-

fitting analysis of responses in equi-

librium for each of the interactions

(lower panels).

FIGURE 2. MS reveals the presence of rRL-6-CH2OH in the E. coli–treated hbMR1 sample. (A) Extracted ion chromatograms (EICs) for rRL-6-CH2-

OH (m/z 329.1103) from hbMR1 protein exposed to E. coli (lower panel) compared with untreated protein control (upper panel). EIC shows compound

with m/z 329.1103 present only in E. coli–treated sample (inset). (B) Compound with m/z 329.1095 (left peak, upper panel) from the E. coli–treated

hbMR1 sample and product ions from targeted fragmentation (lower panel); the structures of each of the products of the fragmentation are shown as insets.

The precursor ion is indicated by a diamond. Tandem mass spectrometry (MS/MS) product ion data match against the theoretical fragmentation pattern of

rRL-6-CH2OH (right peak, upper panel) within ,5 ppm.

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recombinantly expressed, rRL-6-CH2OH–loaded MR1 in crystal-lization screens with the F7 MAIT TCR. Single crystals grownwith these components diffracted well and were used to collecta full data set that refined to 3.3 A (Table I). Overall, the dockingmode of the MAIT TCR is highly similar to that observed withbovine MR1 (9) and human MR1 (16), with the MAIT TCRdocking in a diagonal orientation with respect to the a1 and a2helixes of hbMR1 (Fig. 3A). The a-chain CDR loops of the MAITTCR mostly contact residues of the a2 helix of hbMR1, whereasthe b-chain is biased toward the a1 helix of hbMR1 (Fig. 3B, 3C,Table II). These contacts, with the exception of those made withthe three bovine-specific amino acids (A72, R147, and Q151), areremarkably similar between this complex and our previously re-ported MAIT/bMR1 complex (Supplemental Fig. 1). Moreover,the TCR docking of both of these complexes superimpose almostidentically on the recently published human MAIT/MR1/RL-6-Me-7-OH complex (Fig. 3B), further validating the evolutionaryconservation of MAIT TCR recognition across species. The buriedsurface area of the MAIT TCR–hbMR1 interface is ?1070 A2,with 52 and 48% contributed by the a- and b-chains of the MAITTCR, respectively (Fig. 3C).Previously, we reported a critical role for the CDR3a loop in the

interaction with bMR1; this is confirmed in this complex, withTyr95 of the CDR3a loop positioned directly over the ligand-binding cavity opening. The conformation of the CDR3a loopin this complex is essentially identical to the CDR3a loop con-formations noted in the xenoreactive complex (9) and the unli-ganded human MAIT TCR (15), as well as the human complex(16) (Supplemental Fig. 2), confirming a critical role for Tyr95

in MAIT cell recognition of MR1/ligand and the rigidity of theCDR3a loop upon docking. In addition, we see a role for theCDR3b loop in MR1 recognition due to a subtle shift in confor-mation, resulting in a new contact established with MR1 (G98bwith Trp69 in the hbMR1 a1 helix). As discussed in more detailbelow, the CDR3b loop also makes direct contact with the rRL-6-

CH2OH ligand, demonstrating a clear role for the diverse CDR3bloop in ligand discrimination. Despite the flexibility observed inthe a2 helix in human MR1 (10), the structure with hbMR1 doesnot display evident differences in the backbone positioning, sug-

Table I. Data collection and refinement statistics (molecular replacement)

Data CollectionMAIT TCR F7 MAIT TCR G2 MAIT TCR AE6hMR1-r-RL Bovine MR1 Bovine MR1

Space group P 21 21 21 P 21 21 21 P 21 21 21Cell dimensionsa, b, c (A) 85.9, 88.6, 155.5 83.1, 87.3, 155.8 82.8, 87.0, 156.3a, b, g (˚) 90.00, 90.00, 90.00 90.00, 90.00, 90.00 90.00, 90.00, 90.00Resolution (A) 50–3.3 (3.36–3.3) 50–2.9 (2.95–2.9) 50–3.4 (3.46–3.4)Rmerge 0.072 (0.425) 0.068 (0.7) 0.128 (0.595)I/sI 12.81 (3.92) 20.89 (2.88) 14.76 (5.35)Completeness (%) 97.04 (78.29) 95.79 (93.02) 97.49 (80.54)Redundancy 4.0 (4.1) 6.3 (6.0) 11.4 (9.9)RefinementResolution (A) 3.3 2.9 3.4Total no. reflections 74,945 155,981 179,796No. unique reflections 18,509 27,400 15,806Rwork/Rfree 0.2548/0.3078 0.2433/0.2874 0.2560/0.3105

No. atomsProtein 5954 6288 6020Ligand/ion 43 53 26Water 1 8 1

B factorsProtein 83.70 74.50 91.90Ligand 96.50 102.50 94.90Waters 29.40 52.20 43.20

Root-mean-square deviationsBond length (A) 0.013 0.006 0.008Bond angle (˚) 1.26 0.70 0.99Ramachandran favored (%) 96 96 96Ramachandran outliers (%) 0.28 0 0.41

Data in parentheses are for highest-resolution shell.

FIGURE 3. MAIT TCR recognition of MR1-Ag. (A) Diagram of the

ternary complex structure human MAIT TCR/hbMR1 and the MAIT cell

stimulatory ligand rRL-6-CH2OH. The TCR a- and b-chains are shown in

yellow and brown, respectively; MR1 is cyan; and b2m is teal. rRL-6-

CH2OH is represented as yellow sticks. (B) Superimposition of the F7

MAIT TCR CDR loops in the complexes with bovine (shown in pink and

blue for the a- and b-chains, respectively) and hbMR1 (shown in yellow

and brown for the a- and b-chains, respectively) and comparison with loop

positioning in the human MAIT/MR1/RL-6-Me-7-OH complex (16) (loops

shown in orange and dark blue for the a- and b-chains, respectively). All

three complexes are aligned via MR1, and the respective CDR loops are

displayed on top of the hbMR1 surface. (C) Footprint of the F7 human

MAIT TCR on hbMR1 surface. hbMR1 residues contacting the TCR

a-chain are shown in yellow, and those contacting the b-chain are shown

in brown. Residues making contact with both chains are in pink.

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gesting that hbMR1 does not require special conformational re-arrangements to be engaged by a MAIT TCR (Supplemental Fig. 3).

Slight flexibility was noted in the a2 helix between the humanMR1 unliganded (10) and liganded (16) structures; however, this

Table II. Human F7 MAIT TCR contacts with hbMR1-rRL-6-CH2OH

a-Chain MR1 Contact b-Chain MR1 Contact

CDR1 CDR2Gly28 Glu160 VDW Tyr48 Arg61 VDWPhe29 Glu160 VDW Tyr48 Gln64 VDWPhe29ο Asn155Nd2 Hydrogen bond Ala50 Gln64 VDWPhe29N Glu160Oε1 Hydrogen bond Thr54 Gln64 VDWPhe29ο Glu160Oε1 Hydrogen bond* (3.47) Thr54 Arg67 VDWAsn30 Tyr152 VDW Thr54Og Gln64Nε2 Hydrogen bondAsn30 Asn155 VDW Thr54Og Arg67Nh1 Hydrogen bondAsn30 Trp156 VDW Thr55 Gln64 VDWAsn30 Glu160 VDW Asp56 Gln64 VDWAsn30Nd2 Asn155Nd2 Hydrogen bond* (3.55) CDR3

CDR2 Trp96 Met72 VDWTyr48 His148 VDW Thr97 Trp69 VDWTyr48 Tyr152 VDW Thr97ο Trp69Nε1 Hydrogen bond* (3.65)Val50 Leu151 VDW Gly98 Trp69 VDWVal50 Tyr152 VDW Gly98ο Trp69Nε1 Hydrogen bondVal50 Asn155 VDW Glu99 Glu149 VDWLeu51 Leu151 VDW Gly100 Tyr152 VDWLeu51 Lys154 VDW Ser101Og Glu149Oε1 Hydrogen bondLeu51 Asn155 VDW Ser101N Glu149Oε1 Hydrogen bond* (3.56)Glu55Oε2 His148Nε2 Hydrogen bond Ser101 Glu149 VDWGlu55 His148 VDWArg66 Asn155 VDW b-chain r-RL ContactArg66Nh1 Glu160Oε2 Salt bridge Gly98 OAD,CAJ VDW

CDR3 Glu99 OAD VDWSer93 Glu160 VDW Glu99N OAD Hydrogen bondSer93Og Glu160Oε2 Hydrogen bondSer93 Trp164 VDW MR1 r-RL ContactAsn94Nd2 Arg61Nε HB ( Tyr7 C4A,C4,O4,N3,N5 VDWAsn94 Arg61 VDW Phe8 OAC VDWAsn94 Tyr62 VDW Arg9 CAI,C7,CAJ VDWAsn94Od1 Tyr62OH Hydrogen bond Ser24 OAC,CAI VDWTyr95 Leu65 VDW Ser24Og OAC Hydrogen bondTyr95 Tyr152 VDW Lys43 O4,C4 VDWTyr95 Trp156 VDW Lys43 Nz N3 Hydrogen bond* (3.50)Tyr95OH Tyr152OH Hydrogen bond Lys43 Nz O4 Hydrogen bondTyr95OH Trp156Nε1 Hydrogen bond* (3.73) Tyr62 O2,N3 VDW

Trp69 CAJ,C8A,C4A,N5 VDW

a-chain r-RL Contact Arg94 CAV,C7,CAT VDW

Tyr95 OAF,OAE,CAU VDW Arg94 OAG Hydrogen bond* (3.35)Tyr95OH OAF Hydrogen bond Ile96 OAG VDWTyr95OH OAE Hydrogen bond* (3.60) Tyr152 OAE,OAF VDW

Tyr152 OAE Hydrogen bondGln153 OAG VDWTrp156 OAF,N1,CAK VDW

Hydrogen bonds are $4 A; highly probable hydrogen bonds ($3.3 A) are indicated with an asterisk (*), with the distance shown in parentheses.r-RL, rRL-6-CH2OH

FIGURE 4. Binding of rRL-6-CH2OH in the MR1 pocket and contacts made within MR1 and with the MAIT TCR. (A) Electron density maps for rRL-6-

CH2OH bound in the MR1 groove. Simulated annealing omit map (yellow mesh) and 2F0-FC electron density maps (violet) are displayed and contoured at 1s

together with the ligand, represented as sticks. hbMR1 is shown in cyan, human MR1 in white, rRL-6-CH2OH in yellow, and RL-6-Me-7-OH is shown in white.

Hydrogen bonds are represented by yellow dashed lines. (B) View of rRL-6-CH2OH and the hbMR1 polar and aromatic surrounding residues. (C) Comparison of

binding pocket residues between hbMR1 and human MR1 and positioning of the RL-6-Me-7-OH ligand (RL-Me) reported in (16) (shown in white).

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may be due, in part, to the different resolution of the datasets (3.2A versus 2.0 and 1.9 A).

Structural evidence for the involvement of the ribityl chain inrRL-6-CH2OH recognition by a human MAIT TCR

Electron density for the rRL-6-CH2OH ligand is unambiguous,placing the ligand in a position very similar to that of our model(9) (Fig. 4A) and that observed for 6-FP (9, 10), where the aro-

matic residues lining the cavity interact with the lumazine moiety,primarily through van der Waals (VDW) and p-stacking inter-actions (Fig. 4B). Hydrogen bonds are noted between rRL-6-CH2OH and several MR1 side chains: Ser24, Lys43, Arg94, andTyr152 (Fig. 4A, Table II); they anchor the ligand stably in theligand-binding cavity. The ribityl group extends upward towardthe opening of the ligand-binding cavity, engaging the MAITTCR through both the CDR3a and CDR3b loops. Tyr95 of theCDR3a loop establishes at least one hydrogen bond with theribityl group, as we predicted previously (9). However, in thiscomplex, we see a new role for the CDR3b loop in ligand dis-crimination. A hydrogen bond between the main-chain nitrogenof the CDR3b Glu99 is established with the terminal hydroxyl ofthe ribityl chain of rRL-6-CH2OH. These contacts provide a clearrationale for the enhancement of MAIT cell binding upon rec-ognition of MR1 loaded with stimulatory ligands, such as rRL-6-CH2OH. The conformation of the RL-6-Me-7-OH ligand (16)reported recently in human MR1 is flipped in relation to theconformation of rRL-6-CH2OH reported in this study, resulting inthe ribityl chain of RL-6-Me-7-OH being more sequestered in theMR1-binding cavity (Fig. 4C). This is probably not due to dif-ferences in the MR1-binding pockets, because the positions of theresidues in the two structures superimpose almost perfectly (Fig.4C). The positioning of the RL-6-Me-7-OH ligand in humanMR1 results in only one TCR contact: Tyr95 of the CDR3a loop.This likely is the reason for the reduced functional potency of thisligand in relation to rRL-6-CH2OH (10) and strongly suggeststhat ligands presented by MR1 adopt unique conformations in theligand-binding cavity that are dependent on their chemical struc-ture; these conformations can have direct effects on the functionaloutcome.

FIGURE 5. The CDR3b loop demonstrates conformational flexibility in

ligating hbMR1/rRL-6-CH2OH. The F7 MAIT TCR CDR3b loop bound to

hbMR1/rRL-6-CH2OH is shown in brown. Residues that make VDW and

hydrogen bond contacts are represented as sticks, with the latter denoted

by yellow dashed lines.

FIGURE 6. Three human TCRs

adopt comparable docking modes,

despite different Vb-chain usage.

The CDR loops of the complexes

between G2 MAIT TCR (green) and

AE6 MAIT TCR (purple) with bo-

vine MR1 (cyan) are compared with

the previously reported F7 MAIT

TCR/bovine MR1 complex (TCR in

brown, PDB ID: 4IIQ) (upper left

panel). The complexes were aligned

via the main-chain Ca carbons of

the MR1 H-chain. Details about the

residues that differ between the

TCRs are shown as insets from each

of the complexes: CDR1b (top right

panel). CDR2b (middle right panel).

and CDR3b (bottom panel). Se-

quences of the CDR loops are shown,

and contact residues are underlined.

Hydrogen bonds are represented by

yellow dashed lines.

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Ligand-induced conformational change in CDR3b

To determine whether conformational adjustments play a role inMAIT TCR surveillance of MR1-presented Ags, we compared ourcrystal structure of the F7 MAIT TCR in complex with hbMR1/rRL-6-CH2OH with that of F7 in complex with bovine MR1. Asmall conformational shift of the CDR3b loop was observed,resulting in a hydrogen bond between Gly98b and the Trp69 sidechain of hbMR1 (Figs. 3C, 5). These contacts are remote from thethree species-specific differences noted in bovine MR1; therefore,it is unlikely that the sequence differences are the cause of thisconformational change. Instead, it is more likely that conforma-tional flexibility of the CDR3b loop plays a role in engagement ofcertain MR1-presented stimulatory ligands. As noted previously,the CDR3b loop does not contact the RL-6-Me-7-OH ligand (16),despite having the same amino acid sequence. The overall Cabackbone structure of hbMR1 compared with that of bovine MR1is 1.0 root-mean-square deviation, suggesting that hbMR1 doesnot change substantially upon presentation of a stimulatory ligandor upon engagement of a MAIT TCR (Supplemental Fig. 3).

Variation in MAIT TCR b-chain usage results in uniquecontacts with MR1

We demonstrated previously that variation in the b-chain, eitherthrough Vb domain usage or diversity at the CDR3b loop, can mod-ulate the affinity of the MAIT TCR for MR1 (9). The three MAITTCRs that we examined previously—F7, G2, and AE6 clones—all differ in their CDR3b loop sequences; however, F7 and G2both use Vb13.3 (TRBV6-1) and, therefore, share the same CDR1b

and CDR2b sequences, whereas AE6 uses Vb13.2 (TRBV6-2),which differs in both the CDR1b and CDR2b sequences. F7 andG2 have similar binding affinities to bovine MR1 (30–40 mM), butAE6 binding is ∼2-fold weaker (∼70 mM) (9). This also is reflectedin the measured affinities to hbMR1/rRL-6-CH2OH, where F7 andG2 bind with ∼5 mM affinity, and AE6 is ∼2-fold weaker (8.2 mM)(Fig. 1). To understand the molecular basis for our measured affinitydifference and the role of Vb and CDR3b diversity in MR1 binding,we determined the crystal structures of the G2 and AE6 MAIT TCRsin complex with bovine MR1 to 2.9 and 3.4 A, respectively (Fig. 6,Table I, Supplemental Fig. 4).Comparison of the three xenoreactive structures shows a con-

served docking mode, despite variation in the Vb domain usageand CDR3b loop sequence (Fig. 6). The contacts of the CDRloops from the a-chains are essentially identical, with minorcontact differences attributable to the range of resolution of thesecomplexes. However, variation in contacts of the b-chain weremuch more extensive. In the F7 complex with bovine MR1, nocontact with the CDR1b loop was observed (9), whereas in bothG2 and AE6 the CDR1b loop is involved in MR1 recognition(Fig. 6). In the G2 complex, there is only one VDW contact: Asn30

of CDR1b contacts Gln71 of the MR1 a1 helix. However, the AE6TCR makes more extensive contacts through the CDR1b loop,with three VDW contacts through Glu30 (with Gly68, Gln71, andAla72 of the MR1 a1 helix) and one through Tyr31 with Leu65 ofthe MR1 a1 helix. Glu30 and Tyr31 are amino acid residues uniqueto the Vb13.2 domain, suggesting that Vb-encoded sequence var-iation can play a significant role in MR1 recognition.

Table III. Human G2 MAIT TCR contacts with bovine MR1

a-Chain MR1 Contact b-Chain MR1 Contact

CDR1 CDR1Gly28 Glu160 VDW Asn30 Gln71 VDWPhe29 Glu160 VDW CDR2Phe29ο Asn155Nd2 Hydrogen bond Tyr48 Arg61 VDWPhe29N Glu160Oε1 Hydrogen bond* (3.42) Tyr48 Gln64 VDWPhe29ο Glu160Oε1 Hydrogen bond* (3.60) Ala50 Gln64 VDWAsn30 Tyr152 VDW Ala50 Gly68 VDWAsn30 Trp156 VDW Ser51 Arg67 VDW

CDR2 Ser51 Gly68 VDWTyr48 His148 VDW Glu52 Gln71 VDWTyr48 Tyr152 VDW Thr54 Gln64 VDWVal50 Leu151 VDW Thr54 Arg67 VDWVal50 Tyr152 VDW Thr54Og Gln64Nε2 Hydrogen bondLeu51 Leu151 VDW Thr54Og Gln64Oε1 Hydrogen bondLeu51 Lys154 VDW Thr54Og Arg67Nh1 Hydrogen bondLeu51 Asn155 VDW Thr55 Gln64 VDWGlu55Oε2 Gln151 VDW Thr55ο Gln64Nε2 Hydrogen bondArg66 Asn155 VDW Asp56 Gln64 VDWArg66Nε Asn155Od1 Hydrogen bond* (3.51) CDR3Arg66Nh1 Glu159Oε2 Salt bridge Asp97 Arg61 VDWArg66 Glu159 VDW Asp97 Leu65 VDW

CDR3 Asp97 Trp69 VDWSer93 Glu160 VDW Asp97ο Trp69Nε1 Hydrogen bondAsn94 Tyr62 VDW Pro98 Trp69 VDWAsn94Nd2 Tyr62OH H-bond Asn99Nd2 Glu149Nε1 Hydrogen bondTyr95N Arg61Nh1 H-bond* (3.62)Tyr95 Arg61 VDWTyr95 Leu65 VDWTyr95 Trp69 VDWTyr95 Tyr152 VDWTyr95 Trp156 VDWTyr95OH Tyr152OH Hydrogen bond* (3.44)Tyr95OH Trp156Nε1 Hydrogen bondGln96 Arg61 VDWGln96 Arg61Nε Hydrogen bond* (3.34)

Hydrogen bonds are $4 A; highly probable hydrogen bonds ($3.3 A) are indicated with an asterisk (*), with the distanceshown in parentheses.

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Variable positions in the CDR2b loop between Vb13.3 andVb13.2 are also involved in MR1 binding (Fig. 6). In the F7complex structure, the CDR2b loop has extensive contacts withthe a1 helix of MR1 (9); a highly similar set of contacts is alsoseen in the G2 complex, which shares the same Vb domain(Vb13.3), resulting in main-chain loop conformations that aresuperimposable. AE6, in contrast, establishes fewer contactsthrough the CDR2b loop, and two of the loop-contact residues(Val50 and Ala56) differ from the sequence of F7 and G2 (Ala50

and Asp56). Overall, the AE6 TCR appears to distribute its Vbcontacts over all CDR loops, unlike the CDR2b and CDR3b loopbias observed in F7 and G2.Finally, all three TCRs differ in the amino acid sequences of their

CDR3b loops and adopt different constellations of contacts withMR1. All CDR3b loops bridge the ligand-binding cavity, withcontacts observed between both the a1 and a2 helices of MR1(Fig. 6, lower panel). The CDR3b loop of the F7 TCR is biasedtoward the a2 helix of MR1, with six VDW contacts and twohydrogen bonds. Only four VDW contacts are made with the a1helix of MR1 (9). In contrast, the G2 TCR CDR3b loop estab-lishes the majority of contacts (five of six) with the a1 helix; fourof these are VDW contacts and one, Asp97ο with Trp69Nε1, is a

hydrogen-bond (Table III). Of note is a salt bridge establishedbetween Asp97 of CDR3b and Arg61 of the a1 helix. Asn99 is theonly CDR3b residue that contacts the a2 helix, forming a hydro-gen bond with Glu149Nε1. In the AE6 MAIT TCR clone, thecontacts are distributed between the a1 and a2 helices of MR1and are composed of both hydrogen bonds and VDW interactions(Table IV). Pro97 and Asp98 contact the a1 helix residues Leu65

and Trp69, whereas Gly99 and Gly100 contact His148, Glu149, andTyr152 of the a2 helix.

DiscussionThe evolutionary conservation of the MAIT lineage and themolecule to which they respond suggest that this surveillanceprovides an important function in host protection and/or homeo-stasis. Yet the modulation of MAIT cell reactivity by MR1-presented ligands has remained unclear. The recent identificationof small, ring-based molecules as ligands for MR1 and Ags forMAIT cells (10) expanded our understanding of the signals thatare used to engage the MAIT population and opens up a new classof potential MR1 ligands for MAIT cell modulation. In this study,we provide structural, biochemical, and biophysical data reveal-ing the molecular basis of MAIT cell recognition of an MR1-

Table IV. Human AE6 MAIT TCR contacts with bovine MR1

a-Chain MR1 Contact b-Chain MR1 Contact

CDR1 CDR1Gly28 Glu160 VDW Glu30 Gly68 VDWPhe29 Glu160 VDW Glu30 Gln71 VDWPhe29ο Asn155Nd2 Hydrogen bond Glu30 Ala72 VDWPhe29N Glu160Oε1 Hydrogen bond Tyr31 Leu65 VDWPhe29ο Glu160Oε1 Hydrogen bond* (3.53) CDR2Asn30 Tyr152 VDW Tyr48 Arg61 VDWAsn30 Trp156 VDW Tyr48 Gln64 VDWAsn30 Trp160 VDW Val50 Gln64 VDWCDR2 Val50 Gly68 VDWTyr48 His148 VDW Thr54 Arg67 VDWTyr48 Tyr152 VDW Thr54OH Arg67Nh1 H-bondVal50 Leu151 VDW Thr54 Gln64 VDWVal50 Tyr152 VDW Thr55 Gln64 VDWLeu51 Leu151 VDW Lys67 Gln71 VDWLeu51 Asn155 VDW CDR3Glu55 His148 VDW Pro97 Leu65 VDWGlu55 His148 Hydrogen bond* (3.48) Asp98 Trp69 VDWGlu55 Gln151 VDW Asp98Od1 Trp69 Nε1 Hydrogen bondGly96 Tyr152 VDW Asp98Od2 Trp69Nε1 Hydrogen bondArg66 Asn155 VDW Gly99 Glu149 VDWArg66Nε Asn155Od1 Hydrogen bond* (3.6) Gly99 Tyr152 VDWArg66Nh1 Glu159Oε2 Salt bridge Gly100N His148˚ Hydrogen bond* (3.60)Arg66 Glu159 VDW Gly100˚ His148Nd1 WaterCDR3 Gly100 Tyr152 VDWSer93 Glu160 VDWSer93 Trp164 VDWSer93OH Glu160Oε2 Hydrogen bondAsn94 Tyr62 VDWAsn94 Trp164 VDWAsn94Nd2 Tyr62OH Hydrogen bondTyr95N Arg61Nh1 Hydrogen bond* (3.38)Tyr95 Arg61 VDWTyr95 Leu65 VDWTyr95 Trp69 VDWTyr95 Tyr152 VDWTyr95 Trp156 VDWTyr95OH Tyr152OH Hydrogen bondTyr95OH Trp156Nε1 Hydrogen bond* (3.64)Gln96 Arg61 VDWGln96Nε2 Arg61Nε Hydrogen bond* (3.59)Gln96N Arg61Nh1 Hydrogen bond* (3.57)

Hydrogen bonds are $4 A; highly probable hydrogen bonds ($3.3 A) are indicated with an asterisk (*), with the distanceshown in parentheses.

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presented stimulatory ligand and how diversity in the MAIT cellpopulation via alternative Vb gene usage and CDR3b loop di-versity can further modulate MAIT cell recognition of MR1 li-gand. This work provides a foundation upon which to study thepresentation of other MR1-presented Ags and determine howvariation in the MAIT population translates into Ag recognitionand effector function.Our crystal structure of the F7 MAIT TCR in complex with

hbMR1 loaded with E. coli–derived rRL-6-CH2OH demonstratesa highly conserved docking orientation that is similar to the un-conventional noninvariant NK T TCR–CD1d-sulfatide (18, 19) orclassical ab TCR–MHC–peptide complexes (17), both of whichuse diversity in their CDR3 loops to probe their variable Ags. Thedocking of the TCR onto MR1 is nearly identical to that observedin our ligand-independent, xenoreactive complex (9), as well as therecently described human MAIT/MR1 complex (16), with a re-lated, but different, stimulatory ligand, reinforcing the evolutionaryconservation of this interaction and strongly suggesting that theTCR-docking footprint does not vary with MR1-presented ligands.Our structural data also reveal the orientation of the stimulatory

rRL-6-CH2OH in the hbMR1-binding cavity. Very similar to ourpredicted model and to the placement of the folic acid derivative6-FP (9, 10), rRL-6-CH2OH is surrounded by a cluster of aromaticMR1 residues and hydrogen bonds, with Ser24, Lys43, and Arg94

side chains in the hbMR1 groove. The ribityl chain emerges fromthe MR1 cavity, establishing hydrogen bonds with both theCDR3a and CDR3b loops of the MAIT TCR. These additionalcontacts contributed by Ag to the interaction are consistent withthe enhanced binding of MAIT TCRs with rRL-6-CH2OH–loadedMR1. This orientation is different from that of the RL-6-Me-7-OHligand resolved recently (16) and provides an excellent explana-tion for why this ligand is more stimulatory than RL-6-Me-7-OH.Additional ligand contacts with the CDR3b loop likely enhanceTCR engagement, resulting in the observed enhanced potency.The diversity in ligand conformations also suggests that the MR1-binding cavity can accommodate a range of structures, suggestingthat other small molecules may serve as stimulatory ligands pre-sented by MR1 to MAIT cells.In our hbMR1–rRL-6-CH2OH/MAIT TCR complex, the MAIT

TCR straddles both MR1 a helices, positioning the semi-invariantCDR3a loop and highly diverse CDR3b loop over the opening ofthe MR1 ligand-binding cavity. Observed contacts between bothCDR3 loops and the rRL-6-CH2OH ligand confirm their impor-tance in Ag recognition and suggest that MAIT TCRs use bothconserved amino acid motifs (Tyr95 in the CDR3a loop) and di-versity (CDR3b loop residues) to probe MR1-presented Ags. Theenhancement of MAIT TCR-binding affinity when MR1 presentsrRL-6-CH2OH (20–100-fold enhancement over unloaded MR1)confirms the importance of these ligand contacts in MAIT TCRrecognition of MR1. It is unknown whether the differences in themeasured affinities between MAIT TCRs will dictate the effectorfunction of the cell or whether TCR diversity directs specificity toparticular MR1-presented ligands. However, our demonstrationthat MAIT TCRs can recognize MR1-presented Ags with differentaffinities suggests that MAIT TCR diversity, either through the useof alternate Vb domains or variation within the CDR3b loop,plays an important role in MAIT cell surveillance. Perhaps TCRvariability allows MAIT cells to tune their response to differentMR1-presented Ags, or it may imply alternate functions forMAIT cells in host defense or homeostasis that require a differentaffinity threshold for activation.The complexes between the G2 and AE6 TCRs and bovine MR1

provide additional new insight into the role of b-chain variation inMR1 recognition. Combined with the F7/bMR1 complex, we have

three unique MAIT TCR/MR1 structures for comparison. Thesestructures reveal that variation in the Vb domain can modifycontacts mediated through the CDR1b and CDR2b loops, as seenin the AE6 TCR, which uses Vb13.2 instead of Vb13.3. AE6distributes its Vb contacts over its CDR1 and CDR2 b loops,whereas F7 and G2 contacts are either exclusive to (F7) or heavilybiased toward (G2) the CDR2b loop. Loop-swapping experimentsshowed the importance of the CDR3b loop (15), but our structuresreveal how each TCR establishes a novel constellation of contactswith MR1. Our finding that flexibility in the CDR3b loop plays animportant role in MR1 ligand engagement contrasts with the es-sentially rigid docking of the a-chain CDR loops onto the MR1ligand surface.Future research that extends from this work includes determining

the identity of the MAIT cell–selecting ligand(s) presented by MR1during MAIT cell development in the thymus. How is ligand in-volved in this recognition, or does it merely serve to stabilize MR1expression on the cell surface? Are there endogenous ligands pre-sented by MR1 that are associated with the reported MAIT cellinvolvement in autoimmune disorders (29–31)? Finally, given thereported evidence for the infiltration of Va7.2-Ja33 MAIT cells inkidney and brain tumors (32), are there tumor-derived Ags medi-ating MAIT cell activity in these diseases? Our structures providea molecular model by which ligand presentation by MR1 can bestudied in each of these areas and, importantly, shed light on the roleof MAIT TCR diversity in the engagement of MR1-presented Ags.

AcknowledgmentsWe thank the staff of the General Medicine and Cancer Institutes Collab-

orative Access Team of the Advanced Photon Source (23ID) for the use

of and assistance with X-ray beamlines, particularly Ruslan Sanishvili,

Steven Corcoran, and Michael Becker for help and advice during data

collection. We also sincerely thank Prof. Adelbert Bacher (Institute of

Biochemistry and Food Chemistry, Food Chemistry Division, University of

Hamburg, Hamburg, Germany) for very helpful discussions and assistance

with experimental plans.

DisclosuresThe authors have no financial conflicts of interest.

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