Allosteric disulfide control of ligand binding and endocytosis of the natural killer cell receptor for HLA-G

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 unknown. Random mutagenesis revealed that three cysteines in the first immunoglobulin domain of KIR2DL4 regulated endocytosis and uptake of HLA-G. We found that the Cys10-Cys28 bond visible in the KIR2DL4 crystal structure is an allosteric disulfide with potential to switch to a Cys28-Cys74 bond. Mass spectrometry analysis showed that KIR2DL4 in human cells exists in both disulfide-bonded states. The Cys10-Cys28 bond in purified KIR2DL4 was reduced by protein disulfide isomerase (PDI) in vitro. Inhibition of PDI caused retention of KIR2DL4 at the plasma membrane and prevented HLA-G uptake. 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 endocytosed HLA-G. A structural change predicted by AlphaFold upon disulfide switching to the Cys28-Cys74 form reorients the D0 domain into a conformation that binds HLA-G. Thus, conversion of KIR2DL4 from an inactive state to an HLA-G binding form can regulate NK cell function to promote fetal development.