Identities of P2 and P3 Residues of H-2K^b-Bound Peptides Determine Mouse Ly49C Recognition

<div><p>Ly49 receptors can be peptide selective in their recognition of MHC-I-peptide complexes, affording them a level of discrimination beyond detecting the presence or absence of specific MHC-I allele products. Despite this ability, little is understood regarding the properties that enable some peptides, when bound to MHC-I molecules, to support Ly49 recognition, but not others. Using RMA-S target cells expressing MHC-I molecules loaded with individual peptides and effector cells expressing the ectodomain of the inhibitory Ly49C receptor, we found that two adjacent amino acid residues, P2 and P3, both buried in the peptide binding groove of H-2K^b, determine mouse Ly49C specificity. If both are aliphatic residues, this is supportive. Whereas, small amino acids at P2 and aromatic amino acids at the P3 auxiliary anchor residue are detrimental to Ly49C recognition. These results resemble those with a rat Ly49 where the identity of a peptide anchor residue determines recognition, suggesting that dependence on specific peptide residues buried in the MHC-I peptide-binding groove may be fundamental to Ly49 peptide selectivity and recognition.</p></div

Structural analysis of peptide amino acid docking into the B-pocket of H-2K^b.

<p>The H-2K^b bound peptide amino acid at position 2 (P2) docks into the B-pocket of H-2K^b in H-2K^b-RGYVYQGL. Figure shows the H-2K^b-RGYVYQGL heavy chain in gray ribbon, the RGYVYQGL peptide is in teal. The figure was generated using CHIMERA UCSF software and PDB ID 1KPU for H-2K^b-RGYVYQGL.</p

Structural analysis of peptide amino acid docking into the D-pocket of H-2K^b.

<p>The auxiliary anchor residue at position 3 (P3) docks into the shallow D-pocket of H-2K^b formed by residues within the α2-helix. Comparison of P3 in H-2K^b-SIINFEKL and H-2K^b-RGYVYQGL, complexes that do or do not support Ly49C-H-2K^b interaction, respectively. The figure shows the H-2K^b-SIINFEKL heavy chain in pink ribbon, the SIINFEKL peptide is in dark red; the H-2K^b-RGYVYQGL heavy chain is in gray ribbon, the RGYVYQGL peptide is in teal. The figure was generated using CHIMERA UCSF software and PDB IDs IVAC for H-2K^b-SIINFEKL and 1KPU for H-2K^b-RGYVYQGL.</p

Ly49C peptide dependent recognition of H-2K^b decreases with peptides bearing non-polar aliphatic auxiliary anchor residues.

<p><b>(A)</b> RMA-S cell surface expression of H-2K^b when incubated with the indicated peptides, or no peptide. Shaded histogram corresponds to isotype control. <b>(B)</b> Cytotoxicity assay using RMA-S cells loaded with peptides that have aromatic R groups at P3 co-incubated with RNK.49WC effector cells. <b>(C)</b> Statistically significant changes in Ly49W/C recognition of H-2K^b, with respect to the positive control for RNK.49W/C recognition (SIINFEKL-loaded RMA/S cells), were performed at the 12.5:1 E:T ratio from cytotoxicity assays (*<i>p</i> < 0.05, **<i>p</i> < 0.005). Results plotted are the mean of three independent experiments with error bars representing SD.</p

Peptide residues P2 and P3 determine Ly49C recognition of H-2K^b.

<p><b>(A)</b> Expression of H-2K^b incubated with the indicated peptides. <b>(B)</b> Percent cytotoxicity of RMA-S targets incubated with the indicated peptides incubated with RNK.49W/C effectors. <b>(C)</b> Statistically significant changes in Ly49W/C recognition of H-2K^b, with respect to the positive control for RNK.49W/C recognition (SIINFEKL-loaded RMA/S cells), were conducted at the 12.5:1 E:T ratio from cytotoxicity assays (**<i>p</i> < 0.005). Results plotted are the mean of three independent experiments with error bars representing SD.</p

Sequence and origin of H-2K^b specific peptides bearing non-polar aliphatic auxiliary anchor residues.

<p>Bold indicates primary anchor residue, bold and underlined indicates P3 auxiliary anchor residue.</p><p>Sequence and origin of H-2K^b specific peptides bearing non-polar aliphatic auxiliary anchor residues.</p

Predicted peptide binding affinity to H-2K^b for peptides that poorly, or do not, support Ly49W/C and H-2K^b interaction.

<p>The predicted IC₅₀ values for H-2K^b and specific peptides were obtained using the server NetMHCpan. Predictions were set up to have IC₅₀ value of 10nM for strong affinity and IC₅₀ value of 500nM for weak affinity. In the peptide sequence bold indicates primary anchor residue, bold and underlined indicates P3 auxiliary anchor residue.</p><p>Predicted peptide binding affinity to H-2K^b for peptides that poorly, or do not, support Ly49W/C and H-2K^b interaction.</p

Peptide amino acid substitution demonstrates that auxiliary anchor residue P3 is an important factor in determining Ly49C recognition.

<p><b>(A)</b> Expression of H-2K^b on RMA/S cells co-incubated with the listed peptides <b>(B)</b> Cytotoxic assay using RNK.49W/C effector cells and RMA-S targets incubated with SIINFEKL or the indicated SIINFEKL variants (SIVNFEKL and SIYNFEKL) and RGYVYQGL <b>(C)</b> Statistically significant changes in Ly49W/C recognition of H-2K^b, with respect to the positive control for RNK.49W/C recognition (SIINFEKL-loaded RMA/S cells), were conducted at the 12.5:1 E:T ratio from cytotoxicity assays (*p < 0.05, **p < 0.005). Results plotted are the mean of three independent experiments with error bars indicating SD.</p

Ly49C peptide dependent recognition of H-2K^b is supported using peptides with non-polar aliphatic auxiliary anchor residues.

<p><b>(A)</b> RMA-S cell surface expression of H-2K^b when incubated with the indicated peptides, or no peptide. Shaded histogram corresponds to isotype control. <b>(B)</b> Percent specific lysis of RMA-S cells incubated with peptides bearing aliphatic R groups at P3, in the presence of RNK.49W/C effector cells. <b>(C)</b> Statistically significant changes in Ly49W/C recognition of H-2K^b, with respect to the positive control for RNK.49W/C recognition (SIINFEKL-loaded RMA/S cells), were conducted at the 12.5:1 E:T ratio from cytotoxicity assays (**<i>p</i> < 0.005). Results plotted are the mean of three independent experiments with error bars representing SD.</p

H-2K^b peptide dependent intramolecular interactions that potentially affect H-2K^b and Ly49C association.

<p><b>(A)</b> Comparison between H-2K^b-RGYVYQGL and H-2K^b-SIINFEKL residues that participate in Ly49C interaction. Differences in intramolecular interactions within the H-2K^b that are affected by the peptide bound may be an important factor for the support of receptor and ligand association. Figure shows the H-2K^b-SIINFEKL heavy chain in pink ribbon, the SIINFEKL peptide is in dark red; the H-2K^b-RGYVYQGL heavy chain is in gray ribbon, the RGYVYQGL peptide is in teal. Hydrogen bonds are shown in black dashed lines. <b>(B)</b> Co-crystal structure of H-2K^b-SIINFEKL and Ly49C. Figure shows the H-2K^b-SIINFEKL heavy chain in pink ribbon, the β2m in blue ribbon and the SIINFEKL peptide in dark red. The CTLD of the Ly49C monomer is shown in gold ribbon. The figures were generated using CHIMERA UCSF software and PDB IDs IVAC for H-2K^b-SIINFEKL, 1KPU for H-2K^b-RGYVYQGL and 3C8K for H-2K^b-SIINFEKL-Ly49C.</p