Extensive alanine substitutions increase binding affinity of an influenza nucleoprotein peptide to HLA-Aw68 and do not abrogate peptide-specific CTL recognition

Class I MHC molecules bind peptides in the endoplasmic reticulum and presen t them at the cell surface to circulating CD8(+) T cells for analysis. We h ave examined binding of peptides and stabilization of HLA-Aw68 class I mole cules using synthetic peptide variants of an influenza virus nucleoprotein peptide, NP91-99 (KTGGPIYKR), We have demonstrated that insertion of increa sing numbers of alanines in the center of the peptide (between P and I), to increase a natural bulging out of the peptide-binding cleft, results in a large decrease in thermal stability. Although there is a great decrease in the t(1/2) of the MHC/peptide complex for NP-1A compared with NP91-99, a T cell line, stimulated by NP91-99, recognizes NP-1A efficiently, Peptide var iants with three or more alanines do not show saturable binding to HLA-Aw68 and also are not recognized by the T cell line, Thermal studies show that polyalanine peptides with minimal anchors and nearly all TCR contact residu es exchanged stabilized HLA-Aw68 to high temperatures. Additionally, some o f these polyalanine peptides are recognized by T cell lines generated again st NP91-99. Analysis of the peptide sequences show that the stabilization e ffects are not due to the hydrophobicity of the peptide. These data Suggest that the strength of binding of peptides to HLA-Aw68 is not only dictated by the presence of anchor residues but also by the lack of unfavorable cont acts between the peptide ligand and class I MHC-binding cleft.