{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T16:10:23Z","timestamp":1783959023233,"version":"3.55.0"},"posted":{"date-parts":[[2026,2,19]]},"group-title":"Immunology","reference-count":43,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,19]],"date-time":"2026-02-19T00:00:00Z","timestamp":1771459200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["ZIA AI000525"],"award-info":[{"award-number":["ZIA AI000525"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["RO1 AI170952"],"award-info":[{"award-number":["RO1 AI170952"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"name":"National Health and Medical Research Council of Australia","award":["2008981"],"award-info":[{"award-number":["2008981"]}]},{"name":"National Health and Medical Research Council of Australia","award":["2026631"],"award-info":[{"award-number":["2026631"]}]},{"name":"Heart Foundation Vanguard Grant","award":["110585-2025"],"award-info":[{"award-number":["110585-2025"]}]},{"name":"St George and Sutherland Research Medical Foundation Standard Grant","award":["RG254054"],"award-info":[{"award-number":["RG254054"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  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\n                  <jats:italic>IFI44L<\/jats:italic>\n                  , 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.\n                <\/jats:p>","DOI":"10.64898\/2026.02.18.706601","type":"posted-content","created":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T06:05:19Z","timestamp":1771567519000},"source":"Crossref","is-referenced-by-count":0,"title":["Allosteric disulfide control of ligand binding and endocytosis of KIR2DL4, the natural killer cell receptor for HLA-G"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-7690-2767","authenticated-orcid":false,"given":"Sumati","family":"Rajagopalan","sequence":"first","affiliation":[{"name":"Molecular and Cellular Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, 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USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"George M.","family":"Mastorakos","sequence":"additional","affiliation":[{"name":"Molecular and Cellular Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA"},{"name":"Scienza Health, Inc"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saurav","family":"Majumder","sequence":"additional","affiliation":[{"name":"Molecular and Cellular Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA"},{"name":"Center for Blood Oxygen Transport and Hemostasis, University of Maryland School of Medicine, Baltimore, MD 21201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristof","family":"Nolan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637"},{"name":"AstraZeneca, Inc"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan","family":"Petersen","sequence":"additional","affiliation":[{"name":"Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6271-8574","authenticated-orcid":false,"given":"Erin J.","family":"Adams","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter D.","family":"Sun","sequence":"additional","affiliation":[{"name":"Structural Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jamie","family":"Rossjohn","sequence":"additional","affiliation":[{"name":"Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip J.","family":"Hogg","sequence":"additional","affiliation":[{"name":"Haematology Research Unit, School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia"},{"name":"Centenary Institute, University of Sydney, Camperdown, NSW, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7793-3728","authenticated-orcid":false,"given":"Eric O.","family":"Long","sequence":"additional","affiliation":[{"name":"Molecular and Cellular Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, 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