A novel mechanism of ubiquitin-charging a bivalent E2:RING E3 complex

Ubiquitin, a small protein, is covalently attached to other proteins through a series of enzyme-mediated steps. Initially, an E1 enzyme, activated by binding ubiquitin, facilitates the transfer of ubiquitin from E1 to an E2 enzyme. Subsequently, an E3 enzyme aids in transferring ubiquitin from the charged E2 to a target protein.

RING E3 enzymes, the most abundant E3 family in eukaryotes, serve as scaffolds rather than enzymes, enabling direct ubiquitin transfer from a charged E2 to a protein target through simultaneous engagement with both components. In these RING E3 complexes, an E2 enzyme is positioned at an interface that overlaps with the E1-binding site, and some RING E3s form stable, bivalent complexes with an E2 through additional binding surfaces.

However, how E2 charging occurs within these RING E3 complexes remains a mystery. In a study using human Rad6(Rad18)2 as a model, researchers elucidated the protein-protein interactions among human E1 (Uba1), an E2 (Rad6), and a RING E3 (Rad18) during E2 charging. The findings reveal a novel mechanism distinct from ubiquitin thioester affinity switches.

Instead, the interactions of Rad18 with Rad6 slow the chemistry and preceding steps of the Uba1 catalytic cycle through competitive inhibition, while simultaneously accelerating the release of charged Rad6 from Uba1, ultimately promoting Rad6 charging overall. This marks the first instance of a RING E3 stimulating Uba1-dependent charging of an E2.