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

Abstract

The NK cell inhibitory receptor Ly49A recognizes the mouse MHC class I molecule H-2Dd and participates in the recognition of missing self. Previous studies indicated that the determinant recognized by Ly49A exists in α1/α2 domain of H-2Dd. Here we have substituted polymorphic as well as conserved residues of H-2Dd α1/α2 domain (when compared with H-2Kd, which does not interact with Ly49A). We then tested the ability of the H-2Dd mutants to interact with Ly49A by soluble Ly49A tetramer binding and NK cell cytotoxicity inhibition assays. Individual introduction of mutations converting the H-2Dd residue into the corresponding H-2Kd residue (N30D, D77S, or A99F) in H-2Dd partially abrogated the interaction between Ly49A and H-2Dd. Introduction of the three mutations into H-2Dd completely abolished Ly49A recognition. Individual introduction of D29N or R35A mutation into the residues of H-2Dd that are conserved among murine MHC class I severely impaired the interaction. The crystal structure of H-2Dd 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: α1/α2, α3, and β2-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-specific receptors are sensitive to mutations in MHC class I as well as global loss of MHC class I. Our results also provide insight into the molecular basis of Ly49A to distinguish MHC class I polymorphism.