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

Self Cite

Members of the rodent Ly49 receptor family control NK cell responsiveness and demonstrate allele specificity for MHC class I (MHC-I) ligands. For example, the rat Ly49i2 inhibitory NK cell receptor binds RT1-A1c but not other rat MHC class Ia or Ib molecules. RT1-A1c preferentially binds peptides with proline at the second, or P2, position, which defines it as an HLA-B7 supertype MHC-I molecule. Previously, our laboratory showed that mutations within the MHC-I supertype-defining B-pocket of RT1-A1c could lead to alterations in P2 anchor residues of the peptide repertoire bound by RT1-A1c and loss of recognition by Ly49i2. Although suggestive of peptide involvement, it was unclear whether the peptide P2 anchor residue or alteration of the RT1-A1c primary sequence influenced Ly49i2 recognition. Therefore, we directly investigated the role of the P2 anchor residue of RT1-A1c–bound peptides in Ly49i2 recognition. First, fluorescent multimers generated by refolding soluble recombinant RT1-A1c with individual synthetic peptides differing only at the P2 anchor residue were examined for binding to Ly49i2 NK cell transfectants. Second, cytotoxicity by Ly49i2-expressing NK cells toward RMA-S target cells expressing RT1-A1c bound with peptides that only differ at the P2 anchor residue was evaluated. Our results demonstrate that Ly49i2 recognizes RT1-A1c bound with peptides that have Pro or Val at P2, whereas little or no recognition is observed when RT1-A1c is complexed with peptide bearing Gln at P2. Thus, the identity of the P2 peptide anchor residue is an integral component of MHC-I recognition by Ly49i2.

Section: A1supporting

confidence: 87%

Section: A1mentioning

confidence: 99%

Recognition of Class I MHC by a Rat Ly49 NK Cell Receptor Is Dependent on the Identity of the P2 Anchor Amino Acid of Bound Peptide

Brian

Kane

2011

Self Cite

“…For example, inhibitory KIR3DL1*002, due to a change at residue 238 in one of the extracellular domains, yields stronger levels of inhibition as compared to KIR3DL1*007 when triggered by its HLA-Bw4 ligand (23). Moreover, polymorphic residues affecting the affinity between KIR and its HLA ligands are likely to result in variable degrees of NK licensing and, therefore, effector capability, a phenomenon which has been studied for Ly49A, the murine functional analog of KIR (24, 25). Finally, variation in the KIR extracellular domains has also been demonstrated to affect the levels of surface localization of certain allelic products and, in the case of KIR2DL2*004, the retention of an immature, and likely nonfunctional, isoform within the cytoplasm (26).…”

Section: Introductionmentioning

confidence: 99%

Allelic Variation in KIR2DL3 Generates a KIR2DL2-like Receptor with Increased Binding to its HLA-C Ligand

Frazier

Steiner

Hou

et al. 2013

Although extensive homology exists between their extracellular domains, natural killer cell inhibitory receptors KIR2DL2*001 and KIR2DL3*001 have previously been shown to differ substantially in their HLA-C binding avidity. To explore the largely uncharacterized impact of allelic diversity, the most common KIR2DL2/3 allelic products in European American and African American populations were evaluated for surface expression and binding affinity to their HLA-C group 1 and 2 ligands. Although no significant differences in the degree of cell membrane localization were detected in a transfected human NKL cell line by flow cytometry, surface plasmon resonance and KIR binding to a panel of HLA allotypes demonstrated that KIR2DL3*005 differed significantly from other KIR2DL3 allelic products in its ability to bind HLA-C. The increased affinity and avidity of KIR2DL3*005 for its ligand was also demonstrated to have a larger impact on the inhibition of IFN-γ production by the human KHYG-1 NK cell line compared to KIR2DL3*001, a low affinity allelic product. Site-directed mutagenesis established that the combination of arginine at residue 11 and glutamic acid at residue 35 in KIR2DL3*005 were critical to the observed phenotype. Although these residues are distal to the KIR/HLA-C interface, molecular modeling suggests that alteration in the interdomain hinge angle of KIR2DL3*005 towards that found in KIR2DL2*001, another strong receptor of the KIR2DL2/3 family, may be the cause of this increased affinity. The regain of inhibitory capacity by KIR2DL3*005 suggests that the rapidly evolving KIR locus may be responding to relatively recent selective pressures placed upon certain human populations.

“…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).…”

mentioning

confidence: 99%

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

Mickiewicz

Gays

Lewis

et al. 2014

Ly49B is a potentially important immunoregulator expressed on mouse myeloid cells, and it is thus an unusual member of the wider Ly49 family whose members are ordinarily found on NK cells. Ly49B displays substantial sequence divergence from other Ly49s and in particular shares virtually no amino acid sequence identity with the residues that have been reported to bind to MHC class I (cI) ligands in other Ly49s. Despite this, we show in this study that the BALB/c, but not the C57, isoform of Ly49B displays promiscuous cI binding. Binding was not significantly affected by inactivation of any of the four predicted N-linked glycosylation sites of Ly49B, nor was it affected by removal of the unique 20-aa C-terminal extension found in Ly49B. However, transfer of these C-terminal 20 aa to Ly49A inhibited cI binding, as did the addition of a hemagglutinin tag to the C terminus of Ly49B, demonstrating unexpectedly that the C-terminal region of Ly49s can play a significant role in ligand binding. Systematic exchange of BALB/c and C57 residues revealed that Trp166, Asn167, and Cys251 are of major importance for cI binding in Ly49B. These residues are highly conserved in the Ly49 family. Remarkably, however, Ly49BBALB variants that have C57 residues at positions 166 or 167, and are unable to bind cI multimers, regain substantial cI binding when amino acid changes are made at distal positions, providing an explanation of how highly divergent Ly49s that retain the ability to bind cI molecules might have evolved.