The Structure and Stability of an HLA-A*0201/Octameric Tax Peptide Complex with an Empty Conserved Peptide-N-Terminal Binding Site 1 Amir R. Khan,* Brian M. Baker,* Partho Ghosh, ‡ William E. Biddison, § and Don C. Wiley 2 * ² The crystal structure of the human class I MHC molecule HLA-A2 complexed with of an octameric peptide, Tax8 (LFGYPVYV), from human T cell lymphotrophic virus-1 (HTLV-1) has been determined. This structure is compared with a newly refined, higher resolution (1.8 Å) structure of HLA-A2 complexed with the nonameric Tax9 peptide (LLFGYPVYV) with one more N-terminal residue. Despite the absence of a peptide residue (P1) bound in the conserved N-terminal peptide-binding pocket of the Tax8/ HLA-A2 complex, the structures of the two complexes are essentially identical. Water molecules in the Tax8 complex replace the terminal amino group of the Tax9 peptide and mediate a network of hydrogen bonds among the secondary structural elements at that end of the peptide-binding groove. Thermal denaturation measurements indicate that the Tax8 complex is much less stable, T m  16°C, than the Tax9 complex, but both can sensitize target cells for lysis by some Tax-specific CTL from HTLV-1 infected individuals. The absence of a P1 peptide residue is thus not enough to prevent formation of a “closed conformation” of the peptide-binding site. TCR affinity measurements and cytotoxic T cell assays indicate that the Tax8/HLA-A2 complex does not functionally cross-react with the A6-TCR-bearing T cell clone specific for Tax9/HLA-A2 complexes. The Journal of Immunology, 2000, 164: 6398 – 6405. S hort peptides (9 residues) from proteins degraded in the cytoplasm of vertebrate cells are bound by class I MHC molecules in the endoplasmic reticulum and transported to the cell surface for recognition by the Ag-specific receptors (TCR) of T cells as part of the immune system’s surveillance for foreign Ags (1, 2). Any one class I molecule, of the many different alleles expressed in the population, is capable of forming very stable com- plexes, with half-lives of tens of hours, with a large number of different short peptides. These long-lived MHC/peptide com- plexes, which can mark cells for destruction by CTL, appear to be kinetic traps for peptides. In vitro, the off-rates of peptides from MHC molecules are very slow (3, 4), whereas peptide association rates vary between experimental systems (5–9). The kinetics of MHC assembly suggests a two step process involving a confor- mational change of the MHC molecule from a short-lived, recep- tive, “open” binding state to a long-lived, “closed” conformation (5, 10 –13). X-ray crystal structures of MHC molecules have revealed the structure of the closed conformation with peptides bound (14 –16). Some side chains of bound peptides (anchor residues) are held in pockets in the peptide-binding groove that are polymorphic in the different MHC allelic products, providing a sequence-dependent element to peptide binding (16 –18). In class I MHC molecules the charged N and C termini and main chain of the bound peptide are held, through a network of hydrogen bonds and salt bridges, to polar residues conserved in all human and murine class I MHC molecules (19 –24). This network of conserved hydrogen bonds at both termini of peptides provides an independent peptide se- quence-independent element to peptide binding. Because these peptide N- and C-terminal contacts to class I MHC molecules are conserved for both all bound peptides and all class I alleles, whereas the peptide side chain contacts to the polymorphic pockets of class I molecules vary considerably with different peptides and different MHC alleles, the conserved interactions at the peptide termini have been proposed to have an important role in forming the shared property of long half-life peptide-bound conformations of class I molecules (19). The stability of class I MHC molecules, which are heterodimers of a polymorphic heavy chain (Hc) 3 and  2 -microglobulin ( 2 m), is strongly dependent on the presence of a bound peptide. In vitro, in the absence of bound peptides,  2 m dissociates and Hc aggre- gates (17, 22, 25–29), whereas in vivo in the endoplasmic reticu- lum peptide-free class I molecules are stabilized by chaperonins and the peptide transport and loading proteins, TAP and Tapasin (30, 31). Thermal denaturation studies of MHC/peptide complexes in which either the peptide N-terminal amino group or C-terminal carboxylate group was substituted by a methyl group showed a decrease in the T m of the MHC/peptide of 22°C, indicating a de- crease in stability of 4.6 kcal/mol, whereas substituting both pep- tide anchor residues with alanine showed a decrease of only 5.5°C or 1.2 kcal/mol (28, 32). These thermodynamic data support the suggestion that the conserved interactions between the N and C *Department of Molecular and Cellular Biology and ² Howard Hughes Medical In- stitute, Harvard University, Cambridge MA 02138; ‡ Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093; and § Mo- lecular Immunology Section, Neuroimmunology Branch, National Institute of Neu- rological Disorders and Stroke, National Institutes of Health, Bethesda MD 20892 Received for publication January 28, 2000. Accepted for publication March 30, 2000. 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 A.R.K. is supported by a Human Frontiers Long Term Fellowship. B.M.B. is sup- ported by a fellowship from the Cancer Research Institute. D.C.W. is an investigator of the Howard Hughes Medical Institute. 2 Address correspondence and reprint requests to Dr. Don C. Wiley, Howard Hughes Medical Institute, Department of Molecular and Cellular Biology, Harvard Univer- sity, 7 Divinity Avenue, Cambridge, MA 02138. E-mail address: wiley@xta10. harvard.edu 3 Abbreviations used in this paper: Hc, heavy chain;  2 m,  2 -microglobulin; T m , melting temperature; HTLV-1, human T cell lymphotrophic virus-1; HAM/TSP, HTLV-I-associated myelopathy/tropical spastic paraparesis; CD, circular dichroism. 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