{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:39:08Z","timestamp":1785425948009,"version":"3.56.0"},"posted":{"date-parts":[[2026,7,6]]},"group-title":"Immunology","reference-count":67,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T00:00:00Z","timestamp":1783296000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["1R01AI167646"],"award-info":[{"award-number":["1R01AI167646"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is mediated by the activating IgG receptor CD16a (Fc\u03b3RIIIa), yet the molecular mechanisms governing receptor activation remain poorly understood. We demonstrate that the membrane-distal domain 1 (D1) of CD16a functions as an allosteric checkpoint that controls ADCC independently of IgG-Fc binding. A nanobody, C28, that binds an electronegative patch in D1 dose-dependently blocks NK cell ADCC against multiple therapeutic antibodies without affecting direct cytotoxicity. A second nanobody, C21, binding an adjacent D1 epitope has no such effect. Cryo-EM structures of the CD16a-IgG-nanobody complex reveal that C28 allosterically competes with core-fucosylated IgG and stabilizes a closed D1 conformation resembling unliganded receptor, even when Fc is bound. Molecular dynamics simulations show that occupation of the D1 epitope rigidifies the IgG-binding site, stabilizing CD16a overall in contrast with IgG binding alone. The nanobody C28 restricts CD3\u03b6 phosphorylation in both resting and ADCC-activated NK cells, revealing tonic inhibitory control upstream of the signaling cascade. Using MINFLUX nanoscopy, we also show that CD16a forms dimers of \u223c9 nm spacing on the NK cell surface, a geometry unaltered by the ADCC-enhancing L48H polymorphism. Drawing on structural parallels with the IgE receptor Fc\u03b5RI, which is held inactive as a cholesterol-stabilized dimer, we propose that CD16a dimerization through D1 contacts represents a conserved autoinhibitory mechanism among Fc receptors. Consistent with this model, structure-guided disruption of the C28 epitope in NK-92 cells enhances ADCC potency and killing kinetics, providing a blueprint for engineering improved cellular immunotherapeutics.<\/jats:p>","DOI":"10.64898\/2026.07.03.736431","type":"posted-content","created":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T19:35:16Z","timestamp":1783366516000},"source":"Crossref","is-referenced-by-count":0,"title":["The membrane distal domain of CD16a allosterically regulates NK cell ADCC"],"prefix":"10.64898","author":[{"given":"Tania","family":"Cid","sequence":"first","affiliation":[{"name":"San Diego Biomedical Research Institute, San Diego, CA, USA"},{"name":"Department of Integrative Structural and Computational Biology, Scripps Research, La Jolla, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monica","family":"Fernandez-Quintero","sequence":"additional","affiliation":[{"name":"Department of Integrative Structural and Computational 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