title: Neddylation-induced conformational control regulates cullin RING ligase activity in vivo.
abstract: Cullin RING ligases (CRLs) constitute the largest family of ubiquitin ligases with diverse cellular functions. Conjugation of the ubiquitin-like molecule Nedd8 to a conserved lysine residue on the cullin scaffold is essential for the activity of CRLs. Using structural studies and in vitro assays, it has been demonstrated that neddylation stimulates CRL activity through conformational rearrangement of the cullin C-terminal winged-helix B domain and Rbx1 RING subdomain from a closed architecture to an open and dynamic structure, thus promoting ubiquitin transfer onto the substrate. Here, we tested whether the proposed mechanism operates in vivo in intact cells and applies to other CRL family members. To inhibit cellular neddylation, we used a cell line with tetracycline-inducible expression of a dominant-negative form of the Nedd8 E2 enzyme or treatment of cells with the Nedd8 E1 inhibitor MLN4924. Using these cellular systems, we show that different mutants of Cul2 and Cul3 and of Rbx1 that confer increased Rbx1 flexibility mimic neddylation and rescue CRL activity in intact cells. Our findings indicate that in vivo neddylation functions by inducing conformational changes in the C-terminal domain of Cul2 and Cul3 that free the RING domain of Rbx1 and bridge the gap for ubiquitin transfer onto the substrate.
authors.affiliation: NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, Singapore 117456, Singapore.
authors.forename: Boon Kim
authors.initials: BK
authors.lastname: Boh
authors.forename: Peter G
authors.initials: PG
authors.lastname: Smith
authors.forename: Thilo
authors.initials: T
authors.lastname: Hagen
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044842:Cullin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D000075687:NEDD8 Protein; D011487:Protein Conformation; D034741:RNA, Small Interfering / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
chemical_list: C486317:CAND1 protein, human; C105153:CUL2 protein, human; C503645:CUL3 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C479529:RBX1 protein, human; D034741:RNA, Small Interfering; D014157:Transcription Factors; D025801:Ubiquitin; D014452:Ubiquitins