{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T17:03:17Z","timestamp":1790355797165,"version":"4.1.0"},"posted":{"date-parts":[[2026,1,20]]},"group-title":"Biochemistry","reference-count":54,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T00:00:00Z","timestamp":1768867200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/006w34k90","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,22]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>Melanoma differentiation-associated protein 5 (MDA5), a member of the RIG-I-like receptor family, is a cytoplasmic sensor essential for innate antiviral immunity. MDA5 distinguishes viral RNA from host RNA in part through its ATP hydrolysis activity, which promotes filament turnover on shorter endogenous dsRNAs. Here, we show that the gain-of-function T331I disease-linked mutation within the ATP binding pocket disrupts this balance, resulting in constitutive interferon signaling. Through a combination of cryo-electron microscopy (cryoEM), biochemical assays, and cellular analyses, we reveal the extensive network of interactions that precisely position ATP for catalysis in the wild-type MDA5 ATP binding pocket, and also demonstrate that the T331I mutation impairs ATPase activity, thereby stabilizing MDA5-dsRNA complexes and leading to aberrant immune activation. These findings elucidate how MDA5 ATPase activity regulates antiviral specificity and prevents autoimmunity by controlling filament stability and downstream signaling, offering a mechanistic molecular explanation for disease pathogenesis.<\/jats:p>\n                <jats:sec>\n                  <jats:title>GRAPHICAL ABSTRACT<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"700587v2_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.01.20.700587","type":"posted-content","created":{"date-parts":[[2026,1,21]],"date-time":"2026-01-21T01:15:24Z","timestamp":1768958124000},"source":"Crossref","is-referenced-by-count":0,"title":["Establishing the molecular basis for MDA5 mutation-linked autoimmunity"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9057-0829","authenticated-orcid":false,"given":"Ling","family":"Xu","sequence":"first","affiliation":[{"name":"Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA"},{"name":"Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA"},{"name":"Department of Chemistry, The Wertheim UF Scripps Institute, Jupiter, FL 33458, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1390-4725","authenticated-orcid":false,"given":"Harim","family":"Jang","sequence":"additional","affiliation":[{"name":"Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA"},{"name":"Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0402-6833","authenticated-orcid":false,"given":"Kevin","family":"Chung","sequence":"additional","affiliation":[{"name":"Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rong","family":"Guo","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Yale University, New Haven, CT 06511, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alisia","family":"Pan","sequence":"additional","affiliation":[{"name":"Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9045-8872","authenticated-orcid":false,"given":"Anna Marie","family":"Pyle","sequence":"additional","affiliation":[{"name":"Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA"},{"name":"Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026092509050656000_2026.01.20.700587v2.1","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.103887"},{"key":"2026092509050656000_2026.01.20.700587v2.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tim.2018.08.007"},{"key":"2026092509050656000_2026.01.20.700587v2.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.jhep.2014.11.007"},{"key":"2026092509050656000_2026.01.20.700587v2.4","doi-asserted-by":"crossref","first-page":"718","DOI":"10.1038\/s41419-020-02931-x","article-title":"MDA5 cleavage by the Leader protease of foot-and-mouth disease virus reveals its pleiotropic effect against the host antiviral response","volume":"11","year":"2020","journal-title":"Cell Death Dis"},{"key":"2026092509050656000_2026.01.20.700587v2.5","doi-asserted-by":"crossref","unstructured":"Rebendenne, A. , Valadao, A.L.C. , Tauziet, M. , Maarifi, G. , Bonaventure, B. , McKellar, J. , Planes, R. , Nisole, S. , Arnaud-Arnould, M. , Moncorge, O. et al. 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