The Interaction of KIR3DL1*001 with HLA Class I Molecules Is Dependent upon Molecular Microarchitecture within the Bw4 Epitope

Abstract

The Killer cell Immunoglobulin-like Receptor 3DL1 (KIR3DL1) inhibits activation of Natural Killer (NK) cells upon interaction with Human Leukocyte Antigen (HLA) class I molecules such as HLA-B*57:01 which contain the Bw4 epitope spanning residues 77-83 (e.g. NLRIALR) and not with HLA allomorphs that possess the Bw6 motif (e.g. HLA-B*08:01), which differ at residues 77, 80, 81, 82 and 83. Although Bw4 residues Ile80 and Arg83 directly interact with KIR3DL1*001, their precise role in determining KIR3DL1-HLA-Bw4 specificity remains unclear. Recognition of HLA-B*57:01 by either KIR3DL1+ NK cells or the NK cell line YTS transfected with KIR3DL1*001 was impaired by mutation of residues 80 and 83 of HLA-B*57:01 to the corresponding amino acids within the Bw6 motif. Conversely, the simultaneous introduction of three Bw4 residues at positions 80, 82 and 83 into HLA-B*08:01 conferred an interaction with KIR3DL1*001. Structural analysis of HLA-B*57:01, HLA-B*08:01 and mutants of each bearing substitutions at positions 80 and 83 revealed that Ile80 and Arg83 within the Bw4 motif constrain the conformation of Glu76, primarily through a salt-bridge between Arg83 and Glu76. This salt-bridge was absent in HLA-Bw6 molecules as well as position 83 mutants of HLA-B*57:01. Mutation of the Bw4 residue Ile80 also disrupted this salt-bridge, providing further insight into the role that position 80 plays in mediating KIR3DL1 recognition. Thus the strict conformation of HLA-Bw4 allotypes, held in place by the Glu76-Arg83 interaction, facilitates KIR3DL1 binding whereas Bw6 allotypes present a platform on the α1 helix that is less permissive for KIR3DL1 binding.

A bird's eye view of NK cell receptor interactions with their MHC class I ligands

“…For example, alanine mutations to KIR residues His278 and Leu166 had little impact on binding to HLA‐B*57:01, whereas mutation of the HLA‐B*57:01 residues that these residues contact, Arg83 and Ile80, abrogated binding . Further analyses of the molecular micro‐architecture of the Bw4 motif suggested a salt‐bridge between Arg83 and Glu76 that locked the position of the acidic side‐chain was critical in allowing KIR3DL1 recognition . Notably, in HLA‐Bw6 allotypes this salt‐bridge is absent, as a consequence of the presence of a glycine at position 83, providing an explanation for why KIR3DL receptors do not bind Bw6 allotypes .…”

“…Further analyses of the molecular micro‐architecture of the Bw4 motif suggested a salt‐bridge between Arg83 and Glu76 that locked the position of the acidic side‐chain was critical in allowing KIR3DL1 recognition . Notably, in HLA‐Bw6 allotypes this salt‐bridge is absent, as a consequence of the presence of a glycine at position 83, providing an explanation for why KIR3DL receptors do not bind Bw6 allotypes .…”

“…While the molecular interactions underpinning these changes are unclear, structures of HLA‐B27 and HLA‐B57 have shown that P8 can interact with a number of residues proximal to the Bw4 motif . It is conceivable that the presence of a charged side‐chain at this position may disrupt the internal salt‐bridge between Glu76 and Arg83 of the Bw4 motif, which in turn could reposition Glu76 and impair KIR3DL1 docking .…”

The Structure of the Atypical Killer Cell Immunoglobulin-like Receptor, KIR2DL4

“…Cloning and Expression of HLA-The extracellular domains of HLA-G*01:01 and HLA-B*57:01 (residues 1-275) and ␤ 2 -microglobulin were cloned into the pET-30(b) expression vector for overexpression in E. coli as described previously (31,32). The HLA and ␤ 2 -microglobulin were expressed into inclusion bodies separately in E. coli, refolded, and purified, as described previously (31,32).…”

“…The HLA and ␤ 2 -microglobulin were expressed into inclusion bodies separately in E. coli, refolded, and purified, as described previously (31,32). Briefly, 60 mg of both the HLA-G and HLA-B*57:01 were refolded by rapid dilution in a solution containing 100 mM Tris-HCl, pH 8.0, 400 mM L-arginine-HCl, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione (and 4 M urea for HLA-B*57:01) in the presence of 20 mg ␤ 2 -microglobulin and 10 mg of synthetic peptide.…”