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First, an E1 is activated by attaching ubiquitin to itself through a thioester bond. Next, in E2 charging, activated E1 interacts with an E2 and transfers ubiquitin to a thioester bond on the E2. Finally, an E3 mediates the transfer of ubiquitin from a charged E2 to an isopeptide bond on a protein target. RING E3s, the largest E3 family in eukaryotes, function as scaffolds rather than enzymes and facilitate direct transfer of ubiquitin from a charged E2 to a protein target by simultaneously engaging both. All RING E3s engage an E2 at an interface that overlaps with the E1-binding site and several form stable, bivalent complexes with an E2 through additional binding surfaces. How E2 charging proceeds within these complexes remains unclear. Here, we utilize human Rad6(Rad18)\n                  <jats:sub>2<\/jats:sub>\n                  as a model bivalent E2:RING E3 complex to delineate the interplay of protein\u2022protein interactions among the primary human E1 (Uba1), an E2 (Rad6), and a RING E3 (Rad18) during E2 charging. Collectively, the results reveal a novel mechanism that is not directed by ubiquitin thioester \u201caffinity switches.\u201d Rather, interactions of Rad18 with Rad6 slow the chemistry step and all preceding steps of the Uba1 catalytic cycle via competitive inhibition but accelerate release of charged Rad6 from Uba1, ultimately stimulating Rad6 charging overall. To the best of our knowledge, this represents the first example of a RING E3 stimulating Uba1-dependent charging of an E2.\n                <\/jats:p>","DOI":"10.64898\/2026.09.04.749512","type":"posted-content","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T14:50:11Z","timestamp":1788965411000},"source":"Crossref","is-referenced-by-count":0,"title":["A novel mechanism of ubiquitin-charging a bivalent E2:RING E3 complex"],"prefix":"10.64898","author":[{"given":"Samuel","family":"Perreault","sequence":"first","affiliation":[{"name":"Department of Chemistry, The Pennsylvania State University, University Park, PA 16802"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayush","family":"Mistry","sequence":"additional","affiliation":[{"name":"Department of Biochemistry & Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO 63104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1888-054X","authenticated-orcid":false,"given":"Edwin","family":"Antony","sequence":"additional","affiliation":[{"name":"Department of Biochemistry & Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO 63104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2599-1691","authenticated-orcid":false,"given":"Mark","family":"Hedglin","sequence":"additional","affiliation":[{"name":"Department of Chemistry, The Pennsylvania State University, University Park, PA 16802"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091210100520000_2026.09.04.749512v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.78.101807.093809"},{"key":"2026091210100520000_2026.09.04.749512v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/cr.2016.39"},{"key":"2026091210100520000_2026.09.04.749512v1.3","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111.294975"},{"key":"2026091210100520000_2026.09.04.749512v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb984"},{"key":"2026091210100520000_2026.09.04.749512v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)00057-X"},{"key":"2026091210100520000_2026.09.04.749512v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2011.04.051"},{"key":"2026091210100520000_2026.09.04.749512v1.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2009.10.030"},{"key":"2026091210100520000_2026.09.04.749512v1.8","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biophys-060414-033841"},{"key":"2026091210100520000_2026.09.04.749512v1.9","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1017516108"},{"key":"2026091210100520000_2026.09.04.749512v1.10","doi-asserted-by":"publisher","DOI":"10.1038\/ncb1865"},{"key":"2026091210100520000_2026.09.04.749512v1.11","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkr805"},{"key":"2026091210100520000_2026.09.04.749512v1.12","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444906031003"},{"key":"2026091210100520000_2026.09.04.749512v1.13","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA118.005297"},{"key":"2026091210100520000_2026.09.04.749512v1.14","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkm615"},{"key":"2026091210100520000_2026.09.04.749512v1.15","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/28.14.2847"},{"key":"2026091210100520000_2026.09.04.749512v1.16","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.6b01213"},{"key":"2026091210100520000_2026.09.04.749512v1.17","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.9b00938"},{"key":"2026091210100520000_2026.09.04.749512v1.18","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.0c00849"},{"key":"2026091210100520000_2026.09.04.749512v1.19","doi-asserted-by":"crossref","first-page":"10329","DOI":"10.1016\/S0021-9258(18)34024-9","article-title":"The mechanism of ubiquitin activating enzyme. 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