Distinctive Interactions at Multiple Site 2 Subsites by Allele-Specific Rat and Mouse Ly49 Determine Functional Binding and Class I MHC Specificity

Abstract

Rodent Ly49 exhibit allele-specific MHC I recognition, yet the interaction site, site 2, encompassing the area below the MHC peptide-binding groove, the α3 domain, and associated β2 microglobulin, is highly conserved among rat and mouse MHC I alleles. We previously demonstrated that allele-specific Ly49 recognition can be affected by polymorphisms specifically in the peptide anchor-binding and supertype-defining B pocket of MHC I, possibly through differential conformations assumed by solvent-exposed interaction residues when articulating with this pocket. Through mutagenesis of RT1-A1c and H-2Dd, we map for the first time the interaction site(s) on rat MHC I mediating rat Ly49i2 recognition and the previously unexamined Ly49GBALB/c interaction with H-2Dd. We demonstrate that rat Ly49i2 and mouse Ly49G use both unique and common interactions at three MHC I H chain subsites to mediate functional binding and allele-specific recognition. We find that the F subsite, formed by solvent-exposed residues below the more conserved C-terminal anchor residue-binding F pocket, acts as an anchoring location for both Ly49i2 and Ly49G, whereas these receptors exhibit distinctive reliance on solvent-exposed residues articulating with the polymorphic anchor-binding and supertype-defining pocket(s) at subsite B, as well as on interaction residues at subsite C in the MHC I α3 domain. Our findings, combined with previous Ly49A/H-2Dd and Ly49C/H-2Kb cocrystal data, suggest how allele-specific MHC I conformations and Ly49 polymorphisms may affect Ly49 placement on MHC I ligands and residue usage at site 2, thereby mediating allele-specific recognition at the highly conserved MHC I interface.