Dove, Katja K Stieglitz, Benjamin Duncan, Emily D Rittinger, Katrin Klevit, Rachel E Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs. HHARI;HOIP;Parkin;RBR;ubiquitin E3 ligase;Binding Sites;Catalytic Domain;Humans;Hydrophobic and Hydrophilic Interactions;Models, Molecular;Polycomb Repressive Complex 1;Protein Binding;Protein Conformation;Protein Transport;Tumor Suppressor Proteins;Ubiquitin;Ubiquitin Thiolesterase;Ubiquitin-Conjugating Enzymes;Ubiquitin-Protein Ligases;Ubiquitination;Rittinger FC001142;0601 Biochemistry and Cell Biology;Developmental Biology 2020-07-17
    https://crick.figshare.com/articles/journal_contribution/Molecular_insights_into_RBR_E3_ligase_ubiquitin_transfer_mechanisms/12668552