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.
Department of Biochemistry, University of Washington, Seattle, WA, USA.
Katja K
KK
Dove
Mill Hill Laboratory, The Francis Crick Institute, London, UK.
Benjamin
0000-0003-3634-6997
B
Stieglitz
Department of Biochemistry, University of Washington, Seattle, WA, USA.
Emily D
ED
Duncan
Mill Hill Laboratory, The Francis Crick Institute, London, UK.
Katrin
0000-0002-7698-4435
K
Rittinger
Department of Biochemistry, University of Washington, Seattle, WA, USA klevit@uw.edu.
Rachel E
0000-0002-3476-969X
RE
Klevit
D001665:Binding Sites; D020134:Catalytic Domain; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D063150:Polycomb Repressive Complex 1 / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination
C400134:BAP1 protein, human; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D063150:Polycomb Repressive Complex 1; C080355:RING1 protein, human; D044767:Ubiquitin-Protein Ligases; D043222:Ubiquitin Thiolesterase
HHARI; HOIP; Parkin; RBR; ubiquitin E3 ligase