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

Abstract

Mouse NK cells express Ly-49 receptors specific for classical MHC class I molecules. Several of the Ly-49 receptors have been characterized in terms of function and ligand specificity. However, the only Ly-49 receptor-ligand interaction previously described in detail is that between Ly-49A and H-2Dd, as studied by point mutations in the ligand and the crystal structure of the co-complex of these molecules. It is not known whether other Ly-49 receptors bind MHC class I in a similar manner as Ly-49A. Here we have studied the effect of mutations in Ly-49C on binding to the MHC class I molecules H-2Kb, H-2Db, and H-2Dd. The MHC class I molecules were used as soluble tetramers to stain transiently transfected 293T cells expressing the mutated Ly-49C receptors. Three of nine mutations in Ly-49C led to loss of MHC class I binding. The three Ly-49C mutations that affected MHC binding correspond to Ly-49A residues that are in contact or close to H-2Dd in the co-crystal, demonstrating that MHC class I binding by Ly-49C is dependent on residues in the same area as that used by Ly-49A for ligand contacts.

Reciprocal Transfer of Class I MHC Allele Specificity between Activating Ly-49P and Ly-49W Receptors by Exchange of β4–β5 Loop Residues

“…However, determination of the role of I247 in the differential binding to H-2 s cells in that study was incomplete, because the authors did not further define class I MHC molecules that may be involved, nor did they determine whether the reciprocal mutation, T247I, transferred enhanced binding to H-2 s onto Ly-49I. In another study, Ly-49C residues involved in recognition of multiple class I ligands were identified (30). However, no residues were identified in Ly-49C that discriminated between class I MHC alleles, nor was there acquisition of a new class I specificity, such as for H-2D d , upon mutagenesis of this receptor to selected residues from Ly-49A (30).…”

“…In another study, Ly-49C residues involved in recognition of multiple class I ligands were identified (30). However, no residues were identified in Ly-49C that discriminated between class I MHC alleles, nor was there acquisition of a new class I specificity, such as for H-2D d , upon mutagenesis of this receptor to selected residues from Ly-49A (30). In contrast, our study has demonstrated that the DcGK sequence in the ␤4 -␤5 loop plays an important role in H-2D k allele recognition.…”

“…Despite determination of the Ly-49A-H-2D d cocrystal structure (26) and a number of site-directed mutagenesis studies (25,27,30), the molecular features of Ly-49Rs that confer MHC allele specificity have remained unclear. Using a series of amino acid exchanges between the NK activating receptors Ly-49W and -P, we have determined that three residues, D250, G252, and K253, in the predicted ␤4 -␤5 loop of Ly-49W confer specificity for H-2D k .…”

Critical Residues at the Ly49 Natural Killer Receptor’s Homodimer Interface Determine Functional Recognition of m157, a Mouse Cytomegalovirus MHC Class I-Like Protein

“…In a model where a single m157 molecule is accessed by either Ly49H, Ly49C(S151G), or Ly49U(S151G), the change of dimerization mode introduced by a variation at position 151 would be crucial to confer or abolish recognition to m157. Mutation studies (40,49) and the three-dimensional crystal structures available for Ly49 proteins (21)(22)(23) have revealed a remarkable variability in the manner by which Ly49 inhibitory receptors bind MHC class I. Much of this variation resides in a degree of plasticity at the dimer interface, which imparts notable changes in subunit orientation.…”

Mutagenesis of Ly49B Reveals Key Structural Elements Required for Promiscuous Binding to MHC Class I Molecules and New Insights into the Molecular Evolution of Ly49s

“…Site 2 lacks polymorphic cI residues, providing no explanation for the cI selectivity of Ly49s, and leading to site 1 being originally favored as the principal binding site (12,13). However, most (14)(15)(16)(17)(18)(19), but not all (13,20,21), subsequent mutagenic analyses favored site 2 as the physiological binding site for Ly49s. More recently, the structure of a mutant form of Ly49C in complex with K b revealed a third binding mode in which the Ly49 homodimer adopts a more open dimerization state and contains an additional a helical region in the middle of loop 3, allowing interactions with a slightly different site underneath the peptide-binding groove (site 3) via a single monomer unit (3).…”