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Identification and Structure of an MHC Class I–Encoded Protein with the Potential to Present N-Myristoylated 4-Mer Peptides To Identification and Structure of an MHC Class I−Encoded Protein with the Potential to Present N-Myristoylated 4-mer Peptides 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/ Supplementary http://www.jimmunol.org/content/suppl/2019/04/30/jimmunol.190008 Material 7.DCSupplemental References This article cites 40 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/202/12/3349.full#ref-list-1 by guest on September 28, 2021 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 peptide (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 glycine (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 amino acid 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 ligand 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 infections 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 virus-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 viruses; 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).
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