The Structure of HLA-B8 Complexed to an Immunodominant Viral Determinant: Peptide-Induced Conformational Changes and a Mode of MHC Class I Dimerization 1 Lars Kjer-Nielsen, 2 * Craig S. Clements, 2† Andrew G. Brooks,* Anthony W. Purcell,* Marcos R. Fontes, 3‡ James McCluskey, 4 * and Jamie Rossjohn 4† EBV is a ubiquitous human pathogen that chronically infects up to 90% of the population. Persistent viral infection is charac- terized by latency and periods of viral replication that are kept in check by a strong antiviral CTL response. Despite the size of the EBV genome, CTL immunity focuses on only a few viral determinants but expands a large primary and memory response toward these epitopes. In unrelated HLA-B8  individuals, the response to the immunodominant latent Ag FLRGRAYGL from Epstein Barr nuclear Ag 3A is largely comprised of CTL clones with identical conserved  TCR structures. To better understand the structural correlates of Ag immunodominance and TCR selection bias, we have solved the crystal structure of the HLA-B8- FLRGRAYGL peptide complex to a resolution of 1.9 Å. The structure confirms the importance of P3-Arg, P5-Arg, and P9-Leu as dominant anchor residues involved in peptide binding to HLA-B8. A bulged conformation of the bound peptide provides a structural basis for the critical role of the P7-Tyr residue in T cell recognition. The peptide also induces backbone and side-chain conformational changes in HLA-B8 that are transmitted along the peptide-binding groove in a domino effect. The HLA-B8- FLRGRAYGL complex crystallizes as a dimer in the asymmetric unit and is oriented such that both peptide ligands are projected in the same plane suggesting a higher order arrangement of MHC-peptide complexes that could be involved in formation of the class I Ag-loading complex or in T cell activation. The Journal of Immunology, 2002, 169: 5153–5160. C ontrol of chronic viral infections such as HIV (1), hep- atitis C (2), and EBV (3) requires killing of virus-infected host cells by CTLs that recognize viral peptides pre- sented by HLA class I molecules. A characteristic feature of most antiviral CTL responses is their focus on a limited number of im- munodominant epitopes that together describe the molecular sig- nature of viral replication to killer T cells of the immune system. For instance, the CTL response to EBV in unrelated HLA-B8 + individuals is dominated by reactivity toward the peptide RAK FKQLL from the lytic Ag BZLF1 and FLRGRAYGL (FLR) 5 from the latent Epstein Barr nuclear Ag 3A (4). The biological basis of epitope dominance is complex (5) and can be influenced by the pattern of Ag processing (6, 7), MHC-peptide affinity (8), available T cell repertoire (9), and the constraints of self-tolerance (10). In the CTL response to the HLA-B8-restricted FLR determinant, epitope dominance is further characterized by dominance in the  TCR usage by CTL that recognize this determinant (11). Indeed, the selection of the VDJ combination BV6S2/BSD/BJ2S7 (V6.2/DJ2.7) with the VJ combination AV4S1/AJ14S3 (V4.1/J14.3) occurs repeatedly in FLR-specific CTL from unre- lated individuals with HLA-B8 (11, 12). Moreover, identical res- idues are also encoded by untemplated N region codons present in VDJ and VJ junctional sequences from these FLR-specific CTL. Accordingly, the HLA-B8-FLR response may represent the most extreme example yet of TCR selection bias in an antiviral CTL. This observation has led to the description of these receptors as “public TCRs” (11). These public CTL use mainly germline VDJ and VJ sequences, and their fine specificity is exquisitely sensitive to substitution in the FLR peptide at P4-Gly, P6-Ala, P7-Tyr, and P8-Gly (13). In addition, these T cells also react with allogeneic HLA-B*4402 molecules, presumably complexed to an unknown self peptide (14). In an effort to better understand the structural basis of the immunodominance of FLR in HLA-B8 + individuals and its recognition by a public TCR, we have deter- mined the atomic structure of the HLA-B8-FLR complex at 1.9 Å resolution. The structure confirms the important role of P3-Arg, P5-Arg, and P9-Leu in anchoring the peptide to the HLA-B8 Ag- binding cleft. In addition, P6-Ala, P7-Tyr, and P8-Gly form part of the bulged section of the peptide that results in complete exposure of P7-Tyr, which is accentuated by the small side chains of P6-Ala *Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; † Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia; and ‡ Protein Crystallography Unit, St. Vincent’s Institute of Med- ical Research, 41 Victoria Parade, Fitzroy, Victoria, Australia Received for publication June 19, 2002. Accepted for publication August 23, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 J.R. is supported by a Wellcome Trust Senior Research Fellowship in Biomedical Science in Australia. J.R. also received initial support from St. Vincent’s Institute of Medical Research. A.B. is supported by a R.D. Wright Fellowship. This work was also supported by the National Health & Medical Research Council and the Australian Kidney Foundation. 2 L.K.-N. and C.S.C. contributed equally to this work. 3 Current address: Departamento de Fisica e Biofisica, Instituto de Biociencias, UNESP, Distrito de Rubiao Junior, Botucatu, Sao Paolo, Brazil. 4 Address correspondence and reprint requests to Dr. James McCluskey, University of Melbourne, Department of Microbiology and Immunology, Parkville, Victoria 3010, Australia. E-mail address: jamesm1@unimelb.edu.au; or Dr. Jamie Rossjohn, Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3168, Australia. E-mail address: Jamie.rossjohn@med.monash.edu.au 5 Abbreviations used in this paper: FLR, Epstein Barr nuclear Ag 3 peptide FLR GRAYGL; GGK, HIV-1 Gag protein p17 residues 24 –31 GGKKKYKL; HLA-B8- GGK, HLA-B8-GGK complex; HLA-B8-FLR, HLA-B8-FLR complex; PEG, poly- ethylene glycol; MHCp, MHC-peptide complex; V6.2/DJ2.7, TCR VDJ combination BV6S2/BSD/BJ2S7;V4.1/J14.3, TCR VJ combination AV4S1/ AJ14S3; r.m.s.d., root mean square deviation;  2 m,  2 -microglobulin. The Journal of Immunology Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00