Structural insights into the catalysis and regulation of E3 ubiquitin ligases

Ubiquitin ligases (E3s) recruit ubiquitin-conjugating enzyme (E2)∼ubiquitin (∼ denotes a thioester linkage; here, the linkage is between the catalytic cysteine of E2 and the carboxyl terminus of ubiquitin) complexes and a substrate to promote ubiquitin transfer onto the substrate.The mechanisms of catalysis and regulation of three classes of E3s identified to date — RING (really interesting new gene), HECT (homologous to E6AP C terminus) and RBR (RING-between-RING) — are summarized in this Review.Crystal structures and nuclear magnetic resonance studies have revealed that RING E3s prime E2∼ubiquitin for transfer by promoting a closed E2∼ubiquitin conformation in which the thioester is activated towards nucleophilic attack onto the substrate lysine.Crystal structures of HECT E3s in different stages of catalysis have revealed that conformational changes juxtapose reactants (catalytic cysteine residues of E2 and E3 as well as the catalytic cysteine of E3 and the target residue of the substrate) to prime ubiquitin transfer.Crystal structures have revealed how RBR E3s are autoinhibited in solution. Upon activation, crystal structures of RBR E3s bound to E2∼ubiquitin have shown how ubiquitin is transferred from E2 to E3 and subsequently to the substrate.Substrate lysine selection and the outcome of ubiquitylation depend on spatial arrangements within E2-E3-substrate complexes. Substrate lysine residues that are in proximity to the active sites of E2 or E3 are prioritized for ligation.E3s are regulated by various autoinhibitory mechanisms that hinder their catalytic cycle. Activation frequently requires post-translational modification or binding of protein partners or substrates.