Figure 5. High resolution structures of HLA-B*3501 and HLA-B*3508 presenting APQPAPENAY show that the peptides are presented in different conformations. Structures of APQP complexed to (A) HLA-B*3508 and (B) HLA-B*3501. For clarity, the 2 helix has been removed. 2Fo–Fc electron density, displayed in mesh format, clearly shows the accurate modeling of peptide residues. (C and D) Superposition of the APQP peptides presented by HLA-B*3501 (yellow) and HLA-B*3508 (green) show a dramatic difference in peptide presentation, including (C) a switch from the P5-Ala C group pointing toward the 1 helix (HLA-B*3508) to the 2 helix (HLA-B*3501) and (D) a change from cis–P6-Pro to trans–P6-Pro. closely with that observed in the previously determined HLA-B35 complexes (19, 20). The binding pockets for P2- Pro and P -Tyr, primary anchor residues for HLA-B35, are conserved between this structure and that previously de- scribed (20, 21). Positions P4-Pro, P5-Ala, P6-Pro, P8-Asn, and P9-Ala of the peptide are solvent exposed and thus represent possible TCR contact points. Interestingly, only 21.4% of the 10-mer is solvent exposed when bound to HLA-B*3508, compared with 25% for the previously de- scribed nonameric HLA-B35 complex. This may be attrib- uted to the apparent secondary anchor residue in the HLA- B*3508–APQP complex, where the P7-Glu side chain points down toward the antigen-binding cleft, forming an H bond with Tyr 74 and salt bridging to Arg 97 and the poly- morphic Arg 156. This tethering of the central-bulged re- gion is distinct from several previously determined bulged peptides where the central bulge participates minimally in contacts with the antigen-binding cleft (16, 21). In HLA-B*3508, Arg 156 forms an integral part of an unusual charged cluster of residues, with its guanadinium group stacking antiparallel to the guanadinium group of Arg 97. Interestingly, the high-resolution structure has enabled us to visualize discrete mobility of the Arg 97 and Arg 156 residues, creating two conformations of these residues (Fig. 6 A and Fig. S1, available at http://www.jem.org/cgi/content/ full/jem.20050864/DC1). The Arg 97–Arg 156 interaction 1254 is flanked by two salt-bridging residues: Asp 114, a residue located within the E-pocket, and P7-Glu from the peptide. The extended conformation of the aliphatic moiety of Arg 156 is further stabilized by van der Waals (v.d.w.) interac- tions with Leu 126, Trp 133, and Val 152. In one conforma- tion, Arg 156 also forms a direct H bond with the P3-Gln (Fig. 6 A). The two conformations of Arg 156 and Arg 97 in HLA-B*3508 do not affect the conformation of the solvent- exposed residues of the APQP peptide where no such dis- crete mobility was observed. In HLA-B*3501, the positively charged Arg at position 156 is replaced by the hydrophobic Leu residue that forms v.d.w. contacts with Trp 133, Val 152, and the aliphatic moiety of Asp 114 and unfavorable v.d.w. interactions with the charged Arg 97; Leu 156 does not contact the peptide (Fig. 6 B). To avoid unfavorable interactions with the hy- drophobic Leu 156, the carboxylate moiety of Asp 114 ro- tates away to form a more favorable interaction with Arg 97. Accommodating this polymorphic residue at position 156 requires minimal changes in the HLA-B*3501 heavy chain (the heavy chains in the two complexes are virtually identi- cal; C root mean square deviation 0.25 Å2). However, the polymorphism at position 156 greatly affects the confor- mation of the peptide. Somewhat surprisingly, in the HLA- B*3501–APQP complex, the P7-Glu/Arg 97 salt bridge is maintained despite the loss of the Arg 156 salt bridge. How- THE INFLUENCE OF PEPTIDE CONFORMATION ON A CYTOTOXIC T CELL RESPONSE | Tynan et al.