Impaired MHC class I (H-2Dd)-mediated protection against Ly-49A+ NK cells after amino acid substitutions in the antigen binding cleft

Abstract

The MHC class I molecule H-2Dd (Dd) acts as a ligand for the inhibitory NK cell receptor Ly-49A. We have constructed altered Dd molecules by site-directed mutagenesis, replacing residues with the corresponding amino acids from the Db molecule, which fails to inhibit via Ly-49A. Mutations at positions 73 and 156 (DdS73WD156Y) impaired the protective effect of the Dd molecule, as evaluated by testing lymphoma cells transfected with the mutant gene for sensitivity to killing by Ly-49A+ NK cells in vitro and rejection by NK cells in vivo. The altered residues form a hydrophobic ridge across the floor of the antigen binding cleft. A mutation in the alpha helix of the alpha2 domain, facing the solvent and without direct contact with the peptide (DdA150S) had no effect. Dd recognition by Ly-49A+ NK cells is considered to be peptide dependent, but not peptide specific. Our results indicate that alterations of residues buried in the antigen binding cleft can induce changes in peptide binding patterns and/or conformational changes in the Dd molecule that make the trimolecular complex less permissive for inhibition of Ly-49A+ NK cells.

The NK Cell MHC Class I Receptor Ly49A Detects Mutations on H-2Dd Inside and Outside of the Peptide Binding Groove

“…The important areas include ␤-sheet regions that form the bottom of ␣1/␣2 domain rather than ␣ helices of ␣1/␣2 domains, which form the functional binding site for Ly49A by the crystal structure data and our more recent mutagenesis data (28). Furthermore, other investigators have reported that mutations in residues that affect the peptide binding cleft may also affect Ly49A interaction (29,30). Therefore, the contribution of H-2D d residues that do not directly contact Ly49A requires further investigation.…”

“…Recently, Waldenström et al reported that simultaneous introduction of S73W and D156Y mutation into H-2D d partially impairs H-2D d -mediated protection against killing by Ly49A ϩ NK cells (29). These substitutions putatively introduce a ridge inside the peptide binding groove that is found in H-2D b .…”

“…The functional binding surface is contributed by the three structural domains that constitute H-2D d , ␣1/␣2 and ␣3 domains and ␤ 2 -microglobulin (␤ 2 m), and was referred to as site 2 by Tormo et al (27). Importantly, Asn 30 , Asp 29 , and Arg 25 are at the junction of these three domains and are also in the neighborhood of the functional binding site for Ly49A (Fig. 5).…”

“…In the crystal structure, the COOH-terminal end of ␣1 ␣ helix and the NH 2 -terminal end of ␣2 ␣ helix are engaged by Ly49A with hydrogen bonds at the functional binding site. This structure explains the peptide dependency of Ly49A binding to H-2D d and the sensitivity of the Ly49A binding to the mutations in the peptide binding groove, observed in this and previous studies (29,30).…”

NK Cell Inhibitory Receptor Ly-49C Residues Involved in MHC Class I Binding

“…Site 1 had a relatively small binding area covering the glycosylation site at residue N176, while site 2 had a large binding area spanning the ␣ 1 , ␣ 2 , ␣ 3 , and ␤ 2 -microglobulin (␤ 2 m) domains of H-2D d . Mutational studies of H-2D d have suggested that site 2 is the predominant site of the Ly-49A-H-2D d interaction (22,(32)(33)(34). The Ly-49C-MHC class I interaction has not been studied as extensively.…”