The NK cell MHC class I receptor Ly49A detects mutations on H-2D(d) insideand outside of the peptide binding groove

The NK cell inhibitory receptor Ly49A recognizes the mouse MHC class I mole cule H-2D(d) and participates in the recognition of missing self. Previous studies indicated that the determinant recognized by Ly49A exists in alpha1 /alpha2 domain of H-2D(d). Here we have substituted polymorphic as well as conserved residues of H-2D(d) alpha1/alpha2 domain (when compared with H-2K (d), which does not interact with Ly49A). We then tested the ability of the H-2D(d) mutants to interact with Ly49A by soluble Ly49A tetramer binding a nd NK cell cytotoxicity inhibition assays. Individual introduction of mutat ions converting the H-2D(d) residue into the corresponding H-2K(d) residue (N30D, D77S, or A99F) in H-2D(d) partially abrogated the interaction betwee n Ly49A and H-2D(d). Introduction of the three mutations into H-2D(d) Compl etely abolished Ly49A recognition. Individual introduction of D29N or R35A mutation into the residues of H-2D(d) that are conserved among murine MHC c lass I severely impaired the interaction. The crystal structure of H-2D(d) reveals that D77 and A99 are located in the peptide binding groove and that N30, D29, and R35 are in the interface of the three structural domains of MHC class I: alpha1/alpha2, alpha3, and beta (2)-microglobulin. These data suggest that Ly49A can monitor mutations in MHC class I inside and outside of the peptide binding groove and imply that inhibitory MHC class I-specifi c receptors are sensitive to mutations in MHC class I as well as global los s of MHC class I. Our results also provide insight into the molecular basis of Ly49A to distinguish MHC class I polymorphism.