title: Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B.
abstract: RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.
authors.affiliation: From the Laboratory of Protein Dynamics and Signaling.
authors.forename: Shengjian
authors.initials: S
authors.lastname: Li
authors.affiliation: Macromolecular Crystallography Laboratory, and.
authors.forename: Yu-He
authors.initials: YH
authors.lastname: Liang
authors.affiliation: From the Laboratory of Protein Dynamics and Signaling.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Mariano
authors.affiliation: From the Laboratory of Protein Dynamics and Signaling.
authors.forename: Meredith B
authors.initials: MB
authors.lastname: Metzger
authors.affiliation: From the Laboratory of Protein Dynamics and Signaling.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Stringer
authors.affiliation: From the Laboratory of Protein Dynamics and Signaling.
authors.forename: Ventzislava A
authors.initials: VA
authors.lastname: Hristova
authors.affiliation: Structural Biophysics Laboratory, Center for Cancer Research, NCI, National Institutes of Health, Frederick, Maryland 21702 and.
authors.forename: Jess
authors.initials: J
authors.lastname: Li
authors.affiliation: the Laboratory of Cell and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Randazzo
authors.affiliation: From the Laboratory of Protein Dynamics and Signaling.
authors.forename: Yien Che
authors.initials: YC
authors.lastname: Tsai
authors.affiliation: Macromolecular Crystallography Laboratory, and jix@mail.nih.gov.
authors.forename: Xinhua
authors.initials: X
authors.lastname: Ji
authors.affiliation: From the Laboratory of Protein Dynamics and Signaling, weissmaa@mail.nih.gov.
authors.forename: Allan M
authors.initials: AM
authors.lastname: Weissman
mesh_terms: D006801:Humans; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
chemical_list: C097309:UBE2D2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C476616:RNF25 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: E3 ubiquitin ligase; RING finger; allosteric regulation; crystallography; proteolysis; ubiquitin; ubiquitin-conjugating enzyme (E2 enzyme)