title: Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase.
abstract: RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40-108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12-39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Donald E
authors.initials: DE
authors.lastname: Spratt
authors.affiliation: Department of Oncological Sciences, The Mount Sinai School of Medicine, New York, New York 10029-6574.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Wu
authors.affiliation: Department of Oncological Sciences, The Mount Sinai School of Medicine, New York, New York 10029-6574.
authors.forename: Jordan
authors.initials: J
authors.lastname: Kovacev
authors.affiliation: Department of Oncological Sciences, The Mount Sinai School of Medicine, New York, New York 10029-6574.
authors.forename: Zhen-Qiang
authors.initials: ZQ
authors.lastname: Pan
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada. Electronic address: gshaw1@uwo.ca.
authors.forename: Gary S
authors.initials: GS
authors.lastname: Shaw
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C479529:RBX1 protein, human; D025801:Ubiquitin; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases