A Molecular Basis for How a Single TCR Interfaces Multiple Ligands 1 Alina Boesteanu,* Michael Brehm,* Lawrence M. Mylin,* Gregory J. Christianson, ² Satvir S. Tevethia,* Derry C. Roopenian, ² and Sebastian Joyce 2 * CD8  T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Addi- tionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8  T cells directed against Ags presented by H-2K b class I molecules. When H-2K b contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the K b -binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate. The Journal of Immunology, 1998, 161: 4719 – 4727. T he control of T cell responses by the classical MHC-en- coded Ag-presenting molecules is termed MHC restric- tion (1). MHC restriction entails the presentation of Ag in the form of processed short peptides by the MHC molecules to T cells. MHC class I molecules control the development and function of CD8 + CTL. Class I molecules accomplish their function by chaperoning short intracellular peptides of 8 to 12 aa residues to the cell surface, thus displaying the class I-peptide complexes for an appraisal by circulating CD8 + T cells (reviewed in Ref. 2). A molecular basis for MHC restriction has emerged from solv- ing numerous three-dimensional structures of class I molecules (reviewed in Ref. 3). The heterotrimeric class I molecule consists of the highly diverse heavy chain, the invariant light chain  2 - microglobulin ( 2 m), 3 and a peptide. The 1 and 2 domains of the heavy chain fold in such a manner that they form an Ag- binding groove confined on the sides by two -helices and at the bottom by two -sheets of four antiparallel -strands each. Peptide Ags bind within this groove. The 3 domain of the heavy chain and  2 m attain an Ig-like fold;  2 m is noncovalently associated with the 1, 2, and 3 domains (4, 5). Peptides bind the Ag- binding groove through hydrogen-bonding interactions between the side chain atoms of the conserved residues of pockets A and F of the heavy chain and the main chain atoms of the amino-terminal amine and the carbonyl oxygen of the carboxyl terminus of the peptide, respectively (6 –10). Peptides that bind class I molecules contain structurally conserved residues at the carboxyl terminus (P) and at an additional internal position. A combination of these conserved residues within peptides constitutes the class I binding motif (11–13). Thus H-2K b class I molecules bind peptides that predominantly contain phenylalanine (Phe) or tyrosine (Tyr) at position (P) 5 and a hydrophobic aliphatic residue (e.g., valine (Val), leucine (Leu), and isoleucine (Ile)) at P (11, 12, 14). Pep- tide binding studies using single amino acid variants of antigenic peptides have revealed secondary anchors required for effective binding (15–17). In the case of H-2K b , residues at P2 and P3 serve as secondary anchors (15, 18). The crystal structure of class I molecules also revealed that the majority of the amino acid residues that vary among class I allelic products are those that contact the bound peptide by virtue of their location in the Ag-binding groove. Thus the physico-chemical na- ture of the Ag-binding groove dictates the anchor motif required for specific peptide binding to a class I molecule (5, 19, 20). In the three crystal structures of H-2K b molecules complexed with dif- ferent octameric peptides, the dominant anchors, P5 Phe or Tyr, and the P Leu are completely buried in the groove (8, 9, 21). Of the secondary anchors, those at P2 are tucked into pocket B, and those at P3 are almost completely buried within pocket D of the Ag-binding groove (8, 9, 21), consistent with the view that they can influence peptide binding to H-2K b . Thus, in octameric pep- tides displayed by H-2K b , residues at P2, P3, P5, and P are in intimate contact with the class I molecule; the side chains at the *Department of Microbiology and Immunology, Pennsylvania State University Col- lege of Medicine, Hershey, PA 17033; and ² The Jackson Laboratory, Bar Harbor, ME 04609 Received for publication March 20, 1998. Accepted for publication June 22, 1998. 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 This work was supported by Training Grant CA60395 (M.B.) and by Grants AI34070, CA25000 (S.S.T.), AI28802 (D.C.R.), and HL54977 (S.J.) from the Na- tional Institutes of Health. S.J. is the recipient of the American Cancer Society’s Junior Faculty Research Award. 2 Address correspondence and reprint requests to Dr. Sebastian Joyce, Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, PA 17033. E-mail address: sjoyce@psu.edu 3 Abbreviations used in this paper:  2 m,  2 -microglobulin; CDR, complementarity determining region; gB, glycoprotein B; HSV-I, herpes simplex virus type I; RR-1, ribonucleotide reductase-1. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00