Identification and Structure of an MHC Class I−Encoded Protein with the Potential to Present N-Myristoylated 4-mer to T Cells This information is current as of September 28, 2021. Yukie Yamamoto, Daisuke Morita, Yoko Shima, Akihiro Midorikawa, Tatsuaki Mizutani, Juri Suzuki, Naoki Mori, Takashi Shiina, Hidetoshi Inoko, Yoshimasa Tanaka, Bunzo Mikami and Masahiko Sugita

J Immunol 2019; 202:3349-3358; Prepublished online 1 May Downloaded from 2019; doi: 10.4049/jimmunol.1900087 http://www.jimmunol.org/content/202/12/3349 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identification and Structure of an MHC Class I–Encoded Protein with the Potential to Present N-Myristoylated 4-mer Peptides to T Cells

Yukie Yamamoto,*,† Daisuke Morita,*,† Yoko Shima,*,† Akihiro Midorikawa,*,† Tatsuaki Mizutani,*,† Juri Suzuki,‡ Naoki Mori,x Takashi Shiina,{ Hidetoshi Inoko,{ Yoshimasa Tanaka,‖ Bunzo Mikami,# and Masahiko Sugita*,†

Similar to host proteins, N-myristoylation occurs for viral proteins to dictate their pathological function. However, this lipid- modifying reaction creates a novel class of “lipopeptide” Ags targeted by host CTLs. The primate MHC class I–encoded protein, Mamu-B*098, was previously shown to bind N-myristoylated 5-mer peptides. Nevertheless, T cells exist that recognize even shorter lipopeptides, and much remains to be elucidated concerning the molecular mechanisms of lipopeptide presentation. Downloaded from We, in this study, demonstrate that the MHC class I allele, Mamu-B*05104, binds the N-myristoylated 4-mer (C14-Gly-Gly-Ala-Ile) derived from the viral Nef protein for its presentation to CTLs. A phylogenetic tree analysis indicates that these classical MHC class I alleles are not closely associated; however, the high-resolution x-ray crystallographic analyses indicate that both molecules share lipid-binding structures defined by the exceptionally large, hydrophobic B pocket to accommodate the acylated (G1) as an anchor. The C-terminal isoleucine (I4) of C14-Gly-Gly-Ala-Ile anchors at the F pocket, which is distinct from that of Mamu-B*098 and is virtually identical to that of the peptide-presenting MHC class I molecule, HLA-B51. http://www.jimmunol.org/ The two central residues (G2 and A3) are only exposed externally for recognition by T cells, and the methyl side chain on A3 constitutes a major T cell epitope, underscoring that the epitopic diversity is highly limited for lipopeptides as compared with that for MHC class I–presented long peptides. These structural features suggest that lipopeptide-presenting MHC class I alleles comprise a distinct MHC class I subset that mediates an alternative pathway for CTL activation. The Journal of Immunology, 2019, 202: 3349–3358.

ajor histocompatibility complex class I molecules bind established a paradigmatic model that delineates how peptides are

fragments of intracellular proteins and present them to captured by MHC class I molecules and recognized by TCRs (7, by guest on September 28, 2021 CTLs bearing specific ab TCR and the associated CD8 8). Six pockets, designated A through F, are present in the Ag- M + coreceptors (1, 2). These CD8 CTLs precisely discriminate pep- binding groove of MHC class I molecules; among these pockets, tides derived from self and nonself proteins, thereby monitoring the allele-specific B and F pockets play a major role in influ- microbial insults and cellular transformation that may occur within encing the repertoire (9). The peptides of a stretch of 9 aa cells. As a consequence, CTLs function efficiently in eliminating residues are typically captured with their P2 and C-terminal abnormal cells only while leaving healthy cells unaffected and thus (P9) anchors accommodated in the B and F pockets, respec- serve as a critical element in controlling viral and cancer tively, whereas the side chains of several other residues protrude (3–5). Because of the milestone discovery of the x-ray crystallo- externally for close interactions with TCRs. Accordingly, vir- graphic structure of the peptide-bound HLA–A2 complex (6), ex- tually innumerable T cell epitopic variations may be generated tensive investigations have been conducted over the past three within MHC class I–presented peptides, which allows T cells to decades to elucidate the molecular mechanisms responsible for recognize foreign Ags specifically without eliciting autoimmu- peptide presentation by various MHC class I alleles and have nity. However, this fundamental paradigm may now need some

*Laboratory of Cell Regulation, Institute for Frontier Life and Medical Sciences, (to D.M.) and by a grant from Kato Memorial Bioscience Foundation (to D.M.). It Kyoto University, Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; was also supported by the Cooperation Research Program of the Primate Research †Laboratory of Cell Regulation and Molecular Network, Graduate School of Bio- Institute, Kyoto University. studies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan; The atomic coordinates and structural factors have been submitted to the Protein Data ‡Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto x Bank (https://pdbj.org) under accession numbers 6IWG and 6IWH. University, Inuyama, Aichi 484-8506, Japan; Laboratory of Chemical Ecology, Di- vision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Address correspondence and reprint requests to Prof. Masahiko Sugita, Laboratory of Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan; {Department of Mo- Cell Regulation, Institute for Frontier Life and Medical Sciences, Kyoto University, lecular Life Science, Division of Basic Medical Science and Molecular Medicine, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address: Tokai University School of Medicine, Isehara, Kanagawa 259-1143, Japan; ‖Center [email protected] for Bioinformatics and Molecular Medicine, Graduate School of Biomedical Sci- The online version of this article contains supplemental material. ences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; and #Lab- oratory of Applied Structural Biology, Division of Applied Life Sciences, Graduate Abbreviations used in this article: C14nef4, C14-Gly-Gly-Ala-Ile; C14nef5, C14- School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan Gly-Gly-Ala-Ile-Ser; b2m, b2-microglobulin; RMSD, root mean-square deviation; VDW, van der Waals. ORCIDs: 0000-0002-7011-0745 (Y.Y.); 0000-0002-5176-2561 (H.I.); 0000-0002- 5024-0614 (Y.T.). Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 Received for publication January 22, 2019. Accepted for publication April 11, 2019. This work was supported by Japan Society for the Promotion of Science KAKENHI Grants 17H05791, 18K19563, 18H02852, and 19H04805 (to M.S.), and 16K19151 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900087 3350 STRUCTURE OF LIPOPEPTIDE-PRESENTING MHC CLASS I MOLECULES modifications to incorporate the novel MHC class I function of cDNA was synthesized from 0.5 mg of total RNA using oligo(dT) and the lipopeptide Ag presentation. PrimeScript reverse transcriptase (Takara Bio, Otsu, Japan). PCR ampli- A group of cellular proteins with the N-terminal Gly-x-x-x-Ser/ fication was performed with Pfu DNA polymerase (Stratagene, La Jolla, CA) for 35 cycles at 94˚C for 45 s, at 58˚C for 45 s, and at 72˚C for 1.5 min, Thr motif (where x is any amino acid) undergo N-myristoylation, followed by an additional 10 min incubation at 72˚C. The primers used were a protein lipidation reaction in which N-myristoyltransferase as follows: 59-TAT GGT ACC ATG GCG CCC CGA ACC CTC CTT-39 catalyzes the conjugation of a 14-carbon fatty acid (myristic (sense) and 59-TAT GCG GCC GCC ACA AGA CAG TTG TCT TTT acid) to the N-terminal glycine residue, using myristoyl-CoA as CA-39 (antisense) for Mamu-B*05104 and 59-GCG GAA TTC GAG ACG CCA AGA TGC GGT-39 (sense) and 59-GCG CTC GAG TCA AGC its substrate (10). Besides host proteins, N-myristoylation also CGT GAG AGA CAC AT-39 (antisense) for Mamu-B11L*0101. Mamu-B*06004 occurs for viral proteins in -infected cells by borrowing the cDNA was chemically synthesized (Integrated DNA Technologies). All host machinery (11), and this lipid modification often dictates cDNA samples were cloned into pcDNA3.1(+), and their identity was their pathogenic function (12–14). Therefore, the ability of CTLs confirmed by DNA sequencing. to monitor the N-myristoylation of viral proteins may be valu- T cell assays able for the efficient control of pathogenic ; the findings of our recent studies using SIV-infected monkeys indicated that SN45-derived TCR a and b cDNAs were cloned into pREP7 and pREP9, respectively, and transfection into TCRb-deficient J.RT3 cells was per- T cells capable of mediating such functions exist (15). The formed by electroporation as described previously (21). Cells were cul- + rhesus macaque CD8 CTL line, 2N5.1, specifically recognized tured in the presence of G418 (1.5 mg/ml) and hygromycin B (0.5 mg/ml) N-myristoylated 5-mer peptides (C14-Gly-Gly-Ala-Ile-Ser for the selection of transfectants and used as responder cells in T cell 4 [C14nef5]) derived from the SIV Nef protein, and the classical assays. T cells (5 3 10 /well) were cultured with HeLa cell trans- fectants (5 3 104/well) expressing each Mamu-B allele in the presence of MHC class I–encoded protein, Mamu-B*098, was found to bind Downloaded from 10 mg/ml lipopeptides and 20 nM PMA in 96-well flat-bottom microtiter C14nef5 and functioned as the restriction element for its presen- plates. After 24 h, culture supernatants were collected, and the amount of tation to 2N5.1 (16, 17). IL-2 released into the medium was measured using the BD ELISA kit (BD An x-ray crystallographic analysis of the Mamu-B*098:C14nef5 Biosciences, Franklin Lakes, NJ). complex revealed that the myristoyl group of C14nef5 was ac- Generation of lipopeptide-bound Mamu-B*05104 complexes commodated in the B pocket lined with hydrophobic amino acid Recombinant proteins were prepared as described previously (17). Briefly, residues, whereas the C-terminal residue fitted into the http://www.jimmunol.org/ DNA constructs encoding the ectodomain of Mamu-B*05104 (from G1 to small F pocket. Because the myristoyl group and serine residue at P276 with R128 and K177 mutated into Glu and a Met-Ala added to the the given position are basic elements for most N-myristoylated N terminus) and rhesus b2-microglobulin (b2m) (from I1 to M99 with a proteins (18), Mamu-B*098 may potentially bind a wide array Met-Ala added to the N terminus) were synthesized and cloned into pLM1. of N-myristoylated 5-mer lipopeptides derived not only from the The expression plasmids were introduced into the Escherichia coli Rosetta SIV Nef protein but also from other viral proteins containing 2 (DE3) pLysS strain (Novagen, Madison, WI), and protein expression was induced in the presence of isopropyl-b-D-thiogalactoside, followed by the the N-myristoylation motif. Although 2N5.1 failed to recognize isolation of inclusion bodies. Purified inclusion bodies were dissolved in the N-myristoylated Nef 4-mer lipopeptide (C14-Gly-Gly-Ala-Ile buffer containing 6 M guanidine-HCl, and the insoluble material was re- [C14nef4]) lacking the C-terminal serine residue, the rhesus CD8+ moved by centrifugation. The supernatant was treated with 50 mM DTT at

2 by guest on September 28, 2021 CTL line, termed SN45, isolated from the circulation of a SIV- 37˚C for 3 h, and aliquots were stored at 80˚C until used. C14nef4 and its structural analogue (C14-GGGI), in which A3 was infected donor, exhibited a prominent reactivity to C14nef4 (19). mutated into glycine, were solubilized in methanol. To obtain lipopeptide- Thus, we predicted that another MHC class I allele may exist that loaded Mamu-B*05104 complexes, solubilized Mamu-B*05104 heavy is capable of binding 4-mer lipopeptides in a manner that differs chains (32 mg) and b2m (12 mg) were refolded by rapid dilution in the from that for Mamu-B*098. presence of C14nef4 (7.5 mg) or C14-GGGI (10 mg). After dialysis In the current study, we identified the restriction element for against 10 mM Tris-HCl (pH 8), the refolded proteins were purified by HiLoad 16/600 Superdex 200 pg (GE Healthcare, Milwaukee, WI) size- the presentation of C14nef4 to SN45 as the classical MHC class I exclusion chromatography, followed by monoQ (GE Healthcare) anion allele, Mamu-B*05104. The high-resolution x-ray crystal struc- exchange chromatography. tures of the two MHC class I:lipopeptide complexes (Mamu- B*05104:C14nef4 and Mamu-B*098:C14nef5) revealed marked Crystallization and structural elucidation differences as well as conserved structural features between long Crystals of Mamu-B*05104:lipopeptide complexes were formed as de- peptide- and N-myristoylated short lipopeptide-presenting MHC scribed previously with some modifications (22). Regarding the C14nef4- class I molecules. These structural features indicate that two dis- loaded complex, 1 ml of a 10 mg/ml protein solution and 1 ml of 100 mM MIB buffer (Molecular Dimensions, U.K.) (pH 7) and 25% polyethylene tinct MHC class I subsets have evolved to mediate the presenta- glycol 1500 were mixed at 4˚C. Regarding the C14-GGGI–loaded com- tion of long peptides or short lipopeptides to CTLs. plex, 1 ml of a 10 mg/ml protein solution and 1 ml of a mother liquid containing 200 mM sodium fluoride, 100 mM Bis-Tris propane (pH 7.5), and 20% polyethylene glycol 3350 were mixed at 20˚C. The crystals that Materials and Methods formed were then cryoprotected in 20% ethylene glycol. Diffraction data MHC genotyping and phylogenetic tree analysis were collected at 100 K (in a cold nitrogen gas stream) on a Rigaku Saturn A200 charge-coupled device detector (Rigaku/MSC, Woodlands, TX) us- The typing of Mamu genes was performed as described previously (20). ing synchrotron radiation with a wavelength of 1.0 A˚ at the BL26B1 Briefly, cDNA samples derived from rhesus macaque PBMCs were used as station (SPring-8, Hyogo, Japan). The resulting data set was processed, templates for PCR amplification with primer pairs that were designed to merged, and scaled using HKL-2000 (HKL Research, Charlottesville, amplify all monkey MHC class I genes. Pyrosequencing of the PCR products VA) (23). Complex structures were solved by molecular replacement was performed using the GS Junior system and the amplicon sequencing (MOLREP) with Mamu-B*098 (Protein Data Bank code 4ZFZ) as a search protocol (Roche, Branford, CT). MHC class I genotypes were identified by model, as implemented in CCP4i software (24). Models were refined using referring to the Immuno Polymorphism Database (http://www.ebi.ac.uk/ipd/ the PHENIX software package (25). Structures were rebuilt using COOT index.html). A phylogenetic tree was constructed by the neighbor-joining 0.8.1 (26) and further modified on s-weighted (2|Fo| 2 |Fc|) and (|Fo| 2 |Fc|) method using GENETYX software (GENETYX, Tokyo, Japan) based on the electron density maps. Repeated processes of the rebuilding and refinement a a amino acid sequences of the MHC class I 1/ 2 domains. of Mamu-B*05104:C14nef4 and Mamu-B*05104:C14-GGGI complexes Cloning of MHC class I genes resulted in 96.8 and 98.4% of residues being in favored regions and 0.3 and 0.0% of residues being in outliers, respectively, in a Ramachandran plot. Total RNA was extracted from rhesus macaque (MM570)–derived PBMCs Crystallographic images were visualized using PyMOL (DeLano Scientific, using the RNeasy mini kit (QIAGEN, Hilden, Germany), and first-strand San Carlos, CA). The Journal of Immunology 3351

The root mean-square deviation (RMSD) values over all Ca atoms in the a1 and a2 domains after the superimposition of Mamu-B*05104 with Mamu-A*002 (3JTT) (27), Mamu-B*017 (3RWG) (28), HLA-A2 (3MRE), HLA-B27 (1K5N), HLA-E (3BZE), and HLA-F (5IUE) were calculated. The size of the B pocket, which was lined by amino acid residues at po- sitions 7, 9, 22, 24, 25, 34, 35, 36, 45, 63, 66, 67, 70, 74, 97, and 99, was calculated for each representative MHC class I allele using the CASTp web server (http://cast.engr.uic.edu) (29) with a probe radius of 1.2 A.˚ Accession numbers Atomic coordinates and structural factors for the reported crystal structures have been deposited in the Protein Data Bank (https://pdbj.org) under accession 6IWG (for Mamu-B*05104:C14nef4) and 6IWH (for Mamu- B*05104:C14-GGGI). SN45 TCR tetramer and flow cytometry A soluble form of the SN45 TCR protein was generated using the disulfide- linked TCR method as described previously (30). DNA constructs encoding the extracellular domains of the SN45-derived TCR a-chain (from K1 to G197 with T155 mutated to Cys) and the TCR b-chain (from D1 to D246 with S173 mutated to Cys) that was fused with the C-terminal BirA sub- strate peptide sequence, LHHILDAQKMVWNHR, were cloned into Downloaded from pLM1, and expression plasmids were expressed in E. coli as described above. Purified TCRa (21 mg) and TCRb (35 mg) proteins were combined in 1 l of a buffer containing 100 mM Tris-HCl (pH 8.1), 400 mM L-, 2 mM EDTA, 5 M urea, 3.7 mM cystamine, and 6.6 mM cysteamine and incubated at 10˚C for 24 h with continuous stirring. After dialysis against 6 l of 10 mM Tris-HCl (pH 8), the paired TCR a/b protein was purified as described above, for which intermolecular disulfide bond formation was confirmed by a nonreducing SDS-PAGE analysis. The purified protein was http://www.jimmunol.org/ then biotinylated using the BirA (Sigma-Aldrich), and tetramer A formation was achieved using streptavidin-R–PE (Invitrogen). To assess FIGURE 1. MHC class I alleles expressed in rhesus donors. ( ) Models the reactivity of the TCR tetramer, K562 cell transfectants expressing of the classical MHC class I loci in humans (HLA) and in rhesus macaques Mamu-B*05104 or mock-transfected cells were pulsed with 160 mM (Mamu) are illustrated in parallel. Note that several Mamu-A and Mamu-B lipopeptides for 4 h and then incubated with the TCR tetramer (50 mg/ml) alleles are present per chromosome. (B) Mamu-A and Mamu-B alleles at room temperature for 1 h. Labeled cells were analyzed by flow cytometry expressed in each individual are shown, and those that were shared among using FACS LSRFortessa and FlowJo software. the positive donors and absent in the negative donors were differentially highlighted.

Rhesus macaques (Macaca mulatta) used in the current study were treated by guest on September 28, 2021 humanely in accordance with institutional regulations (31), and experi- production was only observed when T cells were incubated in the mental protocols were approved by the Committee for Experimental Use presence of C14nef4 with HeLa cell transfectants expressing of Nonhuman Primates at the Institute for Frontier Life and Medical Sciences and at the Primate Research Institute of Kyoto University. Mamu-B*05104 but not those expressing Mamu-B*06004 or Mamu-B11L*01 (Fig. 2A, left panel). The C14nef4-specific, Mamu- Results B*05104-dependent response was undetectable for untransfected J.RT3 cells, indicating the involvement of TCRs in Ag recognition Identification of the restriction element for the presentation of (Fig. 2A, right panel). Although Mamu-B*05104 shared sites for C14nef4 to SN45 N- and intradomain disulfide bond formation with The SN45 CTL line, which was established from an SIV-infected Mamu-B*098, significant differences were observed in the primary rhesus monkey (MM521), responded to C14nef4 in the presence of amino acid sequences of their a1 and a2 domains; accordingly, a PBMCs derived from MM521 and three other donors (MM570, phylogenetic tree analysis indicated that Mamu-B*05104 and MM571, and MM606) (“positive donors”), whereas two donors Mamu-B*098, both of which belonged to the classical MHC-B (MM601 and MM1867) failed to activate SN45 (“negative donors”). family, were only remotely associated (Fig. 2B, 2C). Collectively, The clear separation of positive and negative donor groups for these results provide compelling evidence to show that the clas- CTL activation indicated that the presentation of C14nef4 to SN45 sical MHC class I allele, Mamu-B*05104, functions as a novel was mediated by a polymorphic MHC class I allele. The rhesus lipopeptide Ag-presenting molecule capable of mediating the pre- classical MHC class I is marked by the presence of multiple sentation of N-myristoylated 4-mer lipopeptides to CTLs. Mamu-A and Mamu-B alleles per chromosome (32, 33); thus, the expression of several Mamu-A and Mamu-B alleles is simulta- Structure of the C14nef4-bound Mamu-B*05104 complex neously detected in a single individual (Fig. 1A). Deep sequencing The ability of MHC class I molecules to bind N-myristoylated of the MHC class I alleles expressed in each of the donors revealed lipopeptides with a short stretch of 4 aa residues has not been fully that three alleles, Mamu-B*05104, Mamu-B*06004, and Mamu- recognized; therefore, to elucidate the molecular mechanisms B11L*01, were shared among the positive donors and were absent underlying the binding of C14nef4 to Mamu-B*05104, we attempted in the negative donors (Fig. 1B, highlighted in orange, blue, and to clarify the x-ray crystal structure of the Mamu-B*05104:C14nef4 green, respectively). To assess their potential to mediate the pre- complex. The ectodomain of the Mamu-B*05104 H chain and sentation of C14nef4 to SN45, HeLa cell transfectants expressing monkey b2m were produced in E. coli as inclusion bodies, and the each of these alleles were tested for their ability to present C14nef4 purified recombinant proteins were refolded in the presence of to the J.RT3/SN45 responder cells that were obtained by transfec- C14nef4, followed by crystallization. We elucidated the crystal tion of the TCR-deficient J.RT3 T cell line with cDNAs encoding structure of C14nef4-bound Mamu-B*05104 at a resolution of the SN45 TCR a-andb-chains. The J.RT3/SN45 response by IL-2 1.8 A˚ (Table I). The overall structure of Mamu-B*05104 was 3352 STRUCTURE OF LIPOPEPTIDE-PRESENTING MHC CLASS I MOLECULES Downloaded from http://www.jimmunol.org/

FIGURE 2. Identification of Mamu-B*05104 as a lipopeptide-presenting molecule. (A) HeLa cell transfectants expressing Mamu-B*05104, Mamu- B*06004, or Mamu-B11L*01 as well as mock-transfected cells were cultured with SN45 TCR-reconstituted (left panel) or untransfected J.RT3 cells (right panel) in the presence or absence of C14nef4. T cell responses were monitored by measuring the amount of IL-2 released into the medium. Mean values with SEM are shown. (B) A phylogenetic tree constructed by the neighbor-joining method for representative classical (Mamu-A and -B) and nonclassical by guest on September 28, 2021 (Mamu-AG, -I, -E, and -F) MHC class I alleles are shown. Note that Mamu-B*05104 as well as Mamu-B*098 belonged to the classical Mamu-B family. (C) The amino acid sequences of Mamu-B*098 and Mamu-B*05104 are shown, in which unmatched amino acid residues are highlighted. Solid and open triangles indicate paired residues for intramolecular disulfide bonds and the residue for N-glycosylation, respectively. Asterisks indicate amino acid residues that establish VDW interactions with the acyl chain in Mamu-B*098. virtually indistinguishable from that of other MHC class I mole- Accommodation of the myristoyl group in the B pocket cules, in which the two semisymmetrical a1anda2 domains The two anchors, namely, the myristoyl group and C-terminal formed a b-sheet platform topped by two semiparallel a helices isoleucine, located at both ends of the lipopeptide appeared (Fig. 3A) (8). The a1/a2 fold of Mamu-B*05104 exhibited a high to function as bridge piers stabilizing its binding to MHC class I. degree of structural similarity with those of the conven- The acyl chain of C14nef4 extended deeply into the B pocket of tional peptide-presenting MHC class I molecules, including Mamu-B*05104 in a relatively straight configuration, and no al- Mamu-A*002, Mamu-B*017, HLA-A2, and HLA-B27, with ternative conformations were detected. This was apparently dis- ˚ RMSD values of 0.74, 0.53, 0.61, and 0.68 A, respectively, tinct from the U-shaped configuration observed for the acyl chain whereas more structural deviations were noted for the nonclassical of C14nef5 in the B pocket of Mamu-B*098 (Fig. 4A). As was MHC class I molecules (HLA-E and -F) (Fig. 3B). noted previously for the Mamu-B*098 B pocket (Fig. 4A, right Electron density corresponding to C14nef4 was observed in the panel), the B pocket of Mamu-B*05104 was lined with an array of Ag-binding groove of Mamu-B*05104, located between the a1 hydrophobic or noncharged amino acid residues, including Y7, and a2 helices on top of the antiparallel b-sheet (Fig. 3C). Six Y24, V34, F36, T45, A67, W97, and A99, which contacted the pocket structures, designated A through F, that are common to acyl chain of C14nef4 via numerous intermolecular van der MHC class I molecules were also identified in the Ag-binding Waals (VDW) forces (Fig. 4A, left panel). Furthermore, the side groove of Mamu-B*05104 (Fig. 3D); the myristoyl group (C14) chains of R66 and R70 of Mamu-B*05104 were directed outward; and C-terminal isoleucine residue (I4) were buried deeply in the therefore, the polar guanidine groups at their tips were located groove and interacted primarily with the B pocket and F pocket, distantly from the B pocket, whereas their hydrophobic stems respectively, forcing the central 2 aa residues of the peptide chain were able to establish VDW interactions with the acyl chain of to protrude out of the groove and into the solvent (Fig. 3D, 3E). C14nef4 (Fig. 4A, Supplemental Table I). The A pocket of Mamu-B*05104 was unoccupied (Fig. 3D), Besides its hydrophobic properties, the B pocket of Mamu- which contrasted sharply with conventional peptide-presenting B*05104 was also marked by its ample cavity, which was similar MHC class I molecules in which A pockets primarily accommo- to the Mamu-B*098 B pocket. The large cavity size of the two dated the N-terminal residue of peptide ligands (8). lipopeptide-presenting MHC class I alleles appeared exceptional The Journal of Immunology 3353

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

Mamu-B05104:C14nef4a Mamu-B05104:C14-GGGIa Data collection Space group P21 21 21 P21 21 21 Cell dimensions a, b, c (A)˚ 53.82, 81.76, 106.63 55.10 80.79 106.42 a, b, g (˚) 90, 90, 90 90, 90, 90 Resolution (A)˚ a 50–1.80 (1.83–1.80) 50–1.95 (1.98–1.95) Rmerge 0.062 (0.321) 0.073 (0.262) I/sI 43.9 (7.2) 46.0 (10.0) Completeness (%) 99.9 (99.5) 99.8 (98.4) Redundancy 7.1 (6.6) 8.6 (7.8) Refinement Resolution (A)˚ 1.80 (1.84–1.80) 1.95 (2.00–1.95) No. of reflections 44052 (2700) 35237 (2852) Rwork/Rfree (%) 17.9 (19.0)/21.5 (25.7) 17.5 (18.6)/21.7 (26.6) No. of atoms Protein 3133 3115 MYR/sodium/EDO/BO3 15/4/72/12 15/5/52/0 Water 304 255 B-factors (A˚ 2) Downloaded from Protein 25.4 26.4 Ligand and ion 37.8 31.1 Water 31.1 30.8 RMSDs Bond lengths (A)˚ 0.010 0.010 Bond angles (˚) 1.201 1.142

aThe highest resolution shell is shown in parentheses. http://www.jimmunol.org/ EDO, ethylene glycol; MYR, . among MHC class I alleles (higher than 1.5 SD above the mean) amino acid residues at these positions were small for Mamu-B*05104 (Fig. 4B). We found that amino acid residues at positions 9 and 99 (G9 and A99) and Mamu-B*098 (S9 and S99), which provided had a critical influence on the size of the B pocket; in the case of extra space to accommodate the acyl chain (Fig. 4C). conventional peptide-presenting MHC class I alleles, such as Mamu-B*1701 and HLA-B*0801, the side chains of Y9 and Y99 Accommodation of the C-terminal residue in the F pocket and those of D9 and Y99, respectively, protruded into the cavity, The C-terminal isoleucine residue of C14nef4 was accommodated thereby reducing the cavity sizes of the B pockets. In contrast, the in the F pocket of Mamu-B*05104, which was larger than that of by guest on September 28, 2021

FIGURE 3. The overall structure of the Mamu-B*05104:C14nef4 complex. (A) Two views of the trimer complex of the ectodomain of Mamu-B*05104 heavy chains (green), b2m (orange), and C14nef4 (yellow sticks) are shown. (B) Superimposed images of the a1 and a2 domains of Mamu-B*05104 (green) with those of the classical (Mamu-A*00201, -B*1701, HLA-A*0201, and -B*2705) and nonclassical (HLA-E and -F) MHC class I molecules (magenta) are shown with RMSD values provided in parentheses. (C) The binding of C14nef4 (yellow sticks) to Mamu-B*05104 is demonstrated by a 2Fo- Fc map (green mesh) contoured at 0.8s.(D) The surface of the Ag-binding groove with pockets A through F and the bound C14nef4 lipopeptide (yellow sticks) are shown. (E) The bound C14nef4 lipopeptide (yellow sticks) accommodated in the semitransparent Ag-binding groove is shown. 3354 STRUCTURE OF LIPOPEPTIDE-PRESENTING MHC CLASS I MOLECULES Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Interactions with the acyl chain in the B pocket. (A) The C14nef4 lipopeptide (space-filling model) captured in the semitransparent Ag- binding groove of Mamu-B*05104 is shown with the side chains (green) of amino acid residues surrounding the acyl chain (left panel). The C14nef5 lipopeptide captured in Mamu-B*098 is also shown for comparison (right panel). (B) Mamu-B*098, Mamu-B*05104, and other MHC class I molecules for which crystal structures have been resolved are arranged in order of decreasing size of the B pocket cavity. (C) B pockets of Mamu-B*05104 and Mamu- B*098 accommodating the acyl chain (yellow sticks) as well as those of representative peptide-presenting alleles (Mamu-B*1701 and HLA-B*0801) are shown as a semitransparent groove. The side chains of amino acid residues at positions 9 and 99 are also shown with an emphasis on nonbulky side chains at these positions for Mamu-B*05104 and Mamu-B*098.

Mamu-B*098. In the case of Mamu-B*098, the side chain of by establishing VDW interactions with L81, Y84, L95, and Y123 at position 116 protruded into the F pocket, which not of Mamu-B*05104 (Supplemental Table I). only pushed up the bottom of the pocket but also established a unique hydrogen bond with the side chain of the C-terminal serine Potential T cell epitopes residue of C14nef5 (Fig. 5, right panels). Based on these struc- Because the N-myristoylated glycine (C14-G1) and C-terminal tural features, we previously predicted that the Mamu-B*098 isoleucine (I4) residues were submerged deeply in the Ag- F pocket was elaborately designed for binding a small polar amino binding groove of Mamu-B*05104, only the central 2 aa resi- acid residue, such as serine or , which constituted the dues of C14nef4, namely, G2 and A3, were positioned outside of N-myristoylation motif (18). In contrast, the small amino acid the groove (Fig. 3E). Therefore, we attempted to assess the T cell residue at position 116 (S116) of Mamu-B*05104 appeared to be epitopic potential of the methyl group of A3, the sole side chain less influential, making its F pocket more similar to that of con- exposed to the solvent. The SN45 TCR tetramer (Fig. 6A, left), ventional peptide-presenting MHC class I alleles, such as HLA- which was capable of detecting C14nef4 in the context of B51, which had a binding preference for bulky nonpolar residues, Mamu-B*05104, specifically stained K562 cell transfectants including isoleucine (34). We found that the main chain of the expressing Mamu-B*05104 (K562/B*05104) that were pulsed with isoleucine of C14nef4 established a hydrogen bond network with C14nef4 (C14-GGAI) (Fig. 6A, right). In contrast, the tetramer D77, Y84, T143, and K147 of Mamu-B*05104 (Fig. 5, middle failed to react with K562/B*05104 cells pulsed with C14-GGGI, panels), which was virtually identical to that established between the mutant ligand in which the methyl group was removed from A3 HLA-B51 and the C-terminal isoleucine residue of the 9-mer (Fig. 6A, right). Similar results were obtained using J.RT3/SN45 peptide ligand (left panels) (35). The side chain of the C-terminal T cells as responder cells, which recognized C14-GGAI in a dose- isoleucine residue of C14nef4 also contributed to ligand binding dependent manner but totally failed to react to C14-GGGI (Fig. 6B). The Journal of Immunology 3355 Downloaded from

FIGURE 5. Interactions with the C-terminal amino acid residues in the F pocket. Top- and side-view images of F pockets are presented for Mamu-B*05104 (left), HLA-B51 (middle), and Mamu-B*098 (right) as semitransparent grooves with the side chain of the bound C-terminal amino acid residue (isoleucine, isoleucine, and serine, respectively) shown with yellow sticks. Amino acid residues establishing hydrogen bonds (dashed lines)

with the bound C-terminal residue are also indicated. http://www.jimmunol.org/

Furthermore, the high-resolution x-ray crystal structure of were assigned to play different roles in binding peptides and lipo- Mamu-B*05104 complexed with the C14-GGGI mutant lipopeptide peptides. For example, the majority of MHC class I alleles possess (Supplemental Fig. 1, Table I) confirmed that the spatial config- at position 7, and the hydroxyl group at the tip of its side uration of the complex (Fig. 6C) and the hydrogen bond network chain is commonly used to establish a hydrogen bond with the involved in ligand binding (Fig. 6D) were virtually identical to N-terminal amino acid residue of peptide ligands for its anchoring at those observed for the Mamu-B05104:C14nef4 complex, except the A pocket (8). Alternatively, the benzene ring of Y7 was used for the presence or absence of the methyl group of A3. Based on to establish VDW interactions with lipopeptide ligands. Thus, it these functional and structural observations, we concluded that the is interesting to speculate that lipopeptide-presenting MHC class I by guest on September 28, 2021 methyl group of A3 comprised a major epitope recognized by alleles may have emerged during the evolutionary process of SN45 T cells (Fig. 7). polymorphic MHC class I genes, and this is also supported by the F pocket structure and function of Mamu-B*05104 (Fig. 5). As Discussion is the case with HLA-B51 with a binding preference for bulky The MHC class I–mediated presentation of 8–10-mer peptides to nonpolar residues (34), the F pocket of Mamu-B*05104 cap- CTLs is a basic paradigm that modern immunology has estab- tured the Ile residue by establishing numerous VDW interactions lished; however, it is now clear that a couple of MHC class I al- (Supplemental Table I), most of which would be lost if Ser is leles have evolved the ability to present N-myristoylated short replaced for Ile at this position. Therefore, the F pocket of peptide chains to CTLs. Structural analyses of Mamu-B*05104 Mamu-B*05104 is more similar in structure and function to that and Mamu-B*098 highlighted the unique B pocket structure that of HLA-B51 than to that of Mamu-B*098. was distinct from that of conventional peptide-presenting MHC The highly specific recognition of 9-mer peptides by MHC class class I alleles. Amino acid residues that construct the B pocket of I-restricted CTLs is mediated by TCR interactions with multiple MHC class I molecules are highly polymorphic, and this was also epitopic determinants, which generally involve several amino acid the case with the two lipopeptide-presenting MHC class I alleles. residues (7). In contrast, the x-ray crystal structure of the Mamu- A phylogenetic tree analysis indicated that Mamu-B*05104 and B*05104:C14nef4 complex indicated that epitopic variations Mamu-B*098 were not closely associated, and distinct sets of generated within lipopeptide Ags are so limited that the precise amino acid residues constituted the B pockets of Mamu-B*05104 discrimination of self-derived and nonself-derived lipopeptides by (G9, Y24, T45, E63, R66, A67, R70, D74, W97, and A99) and T cells may be almost impossible in a similar manner to that for Mamu-B*098 (S9, V24, M45, Q63, R66, V67, A70, F74, T97, 9-mer peptides. However, SN45 T cells exhibited an exceptional and S99) (Fig. 4A). Nevertheless, these sets of amino acid resi- ability to recognize C14nef4 in the context of Mamu-B*05104 dues functioned similarly to establish VDW interactions with the without showing an apparent autoreactivity to Mamu-B*05104+ acyl chain (Fig. 4A, Supplemental Table I), and the voluminous cells (19). In addition, rhesus monkeys mounted T cell responses B pocket structure was achieved by placing small amino acid to SIV Nef-derived lipopeptide Ags after, but not before, SIV residues at the floor (G9 and A99 for Mamu-B*05104; S9 and , indicating the specific recognition of viral lipopeptides. S99 for Mamu-B*098). These molecular features have not been Results obtained from our preliminary experiments indicated noted previously for peptide-presenting MHC class I alleles, which that a distinct chemical class of self-ligands, such as a phospho- appeared to be more optimal for accommodating the acyl chain lipid species abundantly expressed within cells, comprised a major rather than amino acid residues. ligand repertoire for lipopeptide-presenting MHC class I alleles It is also important to note that some amino acid residues shared in healthy cells. In virus-infected cells, an excess amount of between peptide- and lipopeptide-presenting MHC class I alleles N-myristoylated viral proteins and their degradation products as 3356 STRUCTURE OF LIPOPEPTIDE-PRESENTING MHC CLASS I MOLECULES Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. T cell recognition of bound lipopeptides. (A) The PE-labeled SN45 TCR tetramer (left) was tested for its reactivity to K562 cell transfectants expressing Mamu-B*05104 (K562/B*05104) by flow cytometry (right). Histograms for TCR tetramer-stained cells (filled) and unstained cells (unfilled) are shown. Specific staining was only observed for K562/B*05104 cells that were pulsed with C14nef4 (C14-GGAI) and not those pulsed with the structural analogue, C14-GGGI. (B) HeLa cell transfectants expressing Mamu-B*05104 were cultured with J.RT3/SN45 T cells in the presence of either C14nef4 or C14-GGGI at indicated concentrations. T cell responses were assessed by measuring the amount of IL-2 released into the medium. Mean values with SEM are shown. (C) Top-view images of the a1 and a2 domains of Mamu-B*05104 complexed with C14-GGAI (left, green) or C14-GGGI (middle, pink) are superimposed (right). (D) Hydrogen bond interactions with C14-GGAI (left panel, yellow sticks) as well as those with C14-GGGI (right panel, yellow sticks) are shown as orange dashed lines. Amino acid residues of Mamu-B*05104 that are involved in hydrogen bond interactions are indicated with their side chains displayed as stick models. well as defective ribosomal products may be generated under the long peptides in a highly specific manner may adversely allow influence of inflammatory cytokines (36). Accordingly, the rapid pathogenic viruses to escape simply by mutating a single amino intracellular accumulation of viral lipopeptides may facilitate their acid residue that constitutes the T cell epitope (38). In contrast, presentation to CTLs. the short stretch of N-terminal amino acid residues constitutes Besides the large quantity of CTLs that recognize long peptides, the N-myristoylation motif; therefore, there are few chances for lipopeptide-specific CTLs may constitute a small population; pathogens to introduce arbitrary mutations without affecting the nevertheless, these cells are likely to play a unique role in critical efficiency of N-myristoylation (39, 40). aspects of host defense. N-myristoylation of the Nef protein is Based on the present results, we propose a molecular model essential for its anchoring at the plasma membrane, thereby that recapitulates contrasting features of conventional peptide- exerting its immunosuppressive activity (37). This immune escape presenting and novel lipopeptide-presenting MHC class I subsets mechanism that have evolved may be simultaneously (Fig. 7). N-myristoylated short peptides are captured by MHC counterbalanced by the host immune system through the elicita- class I molecules with their N-terminal C14-Gly and C-terminal tion of lipopeptide-specific CTL responses, which are capable of residues accommodated in the B and F pockets, respectively, sensing the N-myristoylation event in infected cells and elimi- leaving the A pocket unoccupied. The large, hydrophobic nating them. Upon infection, CTLs that recognize Nef-derived B pocket constitutes the most salient feature of lipopeptide- peptides vigorously expand; however, their ability to recognize presenting MHC class I alleles; the F pocket structure appears The Journal of Immunology 3357

FIGURE 7. Molecular models that recapitulate sa- lient features of lipopeptide-presenting MHC class I molecules. Molecular models for lipopeptide-bound Mamu-B*05104 (middle) and Mamu-B*098 (right) are presented with the representative MHC class I allele, HLA-B*51, that binds long peptides (left) for com- parison. Note that the large, hydrophobic B pocket of Mamu-B*05104 is similar to that of Mamu-B*098, whereas its F pocket is virtually identical to that of HLA-B*51. Downloaded from to be variable and may be small (Mamu-B*098) or similar in size 12. Bryant, M., and L. Ratner. 1990. Myristoylation-dependent replication and as- sembly of human immunodeficiency virus 1. Proc. Natl. Acad. Sci. USA 87: (Mamu-B*05104) to that of peptide-presenting MHC class I 523–527. molecules depending on the nature of the C-terminal anchor res- 13. Gripon, P., J. Le Seyec, S. Rumin, and C. Guguen-Guillouzo. 1995. Myr- idue. Because of the short stretch of amino acid residues exposed istylation of the hepatitis B virus large surface protein is essential for viral in- fectivity. Virology 213: 292–299. to the solvent, the T cell epitopic variations generated within 14. Perez, M., D. L. Greenwald, and J. C. de la Torre. 2004. Myristoylation of the lipopeptide ligands are more limited than those generated within RING finger Z protein is essential for arenavirus budding. J. Virol. 78: 11443– http://www.jimmunol.org/ long peptide ligands; however, T cells are capable of monitoring 11448. 15. Morita, D., T. Igarashi, M. Horiike, N. Mori, and M. Sugita. 2011. Cutting edge: the presence or absence of the alkyl side chain of the ligand, in- T cells monitor N-myristoylation of the Nef protein in simian immunodeficiency cluding the methyl group of A3 of C14nef4 captured by Mamu- virus-infected monkeys. J. Immunol. 187: 608–612. 16. Morita, D., and M. Sugita. 2016. Lipopeptides: a novel antigen repertoire pre- B*05104. Structural differences as well as similarities between sented by major histocompatibility complex class I molecules. Immunology 149: long peptide- and short lipopeptide-presenting MHC class I mol- 139–145. ecules are now beginning to be elucidated, providing novel in- 17. Morita, D., Y. Yamamoto, T. Mizutani, T. Ishikawa, J. Suzuki, T. Igarashi, N. Mori, T. Shiina, H. Inoko, H. Fujita, et al. 2016. Crystal structure of the sights into their coevolution. N-myristoylated lipopeptide-bound MHC class I complex. Nat. Commun. 7: 10356. 18. Maurer-Stroh, S., B. Eisenhaber, and F. Eisenhaber. 2002. N-terminal N- by guest on September 28, 2021 Acknowledgments myristoylation of proteins: refinement of the sequence motif and its taxon- We thank Dr. Tatsuhiko Igarashi for his helpful discussions concerning specific differences. J. Mol. Biol. 317: 523–540. experiments. 19. Morita, D., Y. Yamamoto, J. Suzuki, N. Mori, T. Igarashi, and M. 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A B Side view 1 2 1

Gly2 Gly3 3 Gly1

2m C14 Ile4

Supplementary Figure 1. The crystal structure of the Mamu-B*05104:C14-GGGI complex

(A) An X-ray crystallographic structure of the trimer complex of Mamu-B*05104 heavy chains (purple),

β2m (orange), and C14nef4 (yellow sticks) is shown. (B) The binding of C14-GGGI (yellow sticks) to Mamu-

B*05104 is demonstrated by a 2Fo-Fc map (purple mesh) contoured at 0.8σ (upper panel). The bound C14-

GGGI lipopeptide (yellow sticks) accommodated in the semi-transparent antigen-binding groove is shown

(lower panel). Supplementary Table I. Contacts between C14nef4 and Mamu-B*05104

Hydrogen bonds* C14nef4 C-C contacts* Atom Partner Distance (Å) Myristoyl O1 Water265 2.7 Y7, Y24, V25, V34, F36, T45, group Water266 2.8 R66, A67, R70, W97, A99, H114 Gly1 N Y152 (OH) 2.9 T73, Y152 Water247 3.0 O R70 (NH2) 3.2 Gly2 None T73 Ala3 N Water265 3.1 T73, D77 O W147 (NE1) 2.9 Ile4 N D77 (OD1) 2.6 D77, T80, L81, Y84, L95, Y123, Water179 3.2 T143, K146, W147 O Y84 (OH) 3.1 T143 (OG1) 2.7 OXT K146 (NZ) 2.8 Water179 2.5 * Contacts were assigned with a cut-off of 3.2 Å for hydrogen bond interactions and 4.4 Å for C-C interactions.