Allosteric disulfide control of ligand binding and endocytosis of KIR2DL4, the natural killer cell receptor for HLA-G.
Rajagopalan Sumati S, Chiu Joyce J, Chaurasia Priyanka P, Lu Jinghua J et al.
Human Leukocyte Antigen (HLA)-G is selectively expressed by fetal trophoblast cells that invade maternal tissue and encounter maternal natural killer (NK) cells early in pregnancy. In NK cells, the endosomal receptor KIR2DL4 responds to soluble HLA-G by inducing a broad transcriptional program to support placental development. Structural features of KIR2DL4 that control ligand binding and endocytosis are unclear. Random mutagenesis revealed that three cysteines in the first immunoglobulin domain of KIR2DL4 regulate endocytosis and uptake of HLA-G. We found that the atypical Cys10-Cys28 disulfide bond observed in the KIR2DL4 crystal structure is an allosteric disulfide with potential to switch to a conventional Cys28-Cys74 bond. KIR2DL4 in human cells exists in both disulfide-bonded states, as shown by mass spectrometry analysis. The Cys10-Cys28 bond in a Cys74Ser KIR2DL4 mutant was reduced by protein disulfide isomerase (PDI) in vitro. Inhibition of PDI prevented HLA-G uptake by KIR2DL4 in both transfected 293T cells and primary NK cells. Mutants in the Cys10-Cys28 configuration endocytosed spontaneously but did not bind HLA-G. Conversely, KIR2DL4 with a Cys28-Cys74 bond was at the plasma membrane and responded to soluble HLA-G by endocytosis and transcription of the interferon stimulated gene IFI44L , like wild-type cells. A purified Cys10Leu KIR2DL4 mutant, which exhibited a reduced tendency to oligomerize, bound HLA-G in a peptide dependent manner with an affinity in the low micromolar range. Disulfide switching from the Cys10-Cys28 to the Cys28-Cys74 form correlated with a distant allosteric change, as predicted by AlphaFold, that could reorient a D0 domain loop to facilitate HLA-G binding. Thus, conversion from an inactive state to an HLA-G binding form regulates KIR2DL4 cellular localization and function to promote fetal development.