title: Mechanism of ubiquitylation by dimeric RING ligase RNF4.
abstract: Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight into the mechanism of RING-mediated ubiquitylation, we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model, the 'Ile44 hydrophobic patch' of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING, and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis.
authors.affiliation: Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Plechanovová
authors.forename: Ellis G
authors.initials: EG
authors.lastname: Jaffray
authors.forename: Stephen A
authors.initials: SA
authors.lastname: McMahon
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Johnson
authors.forename: Iva
authors.initials: I
authors.lastname: Navrátilová
authors.forename: James H
authors.initials: JH
authors.lastname: Naismith
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Hay
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D020134:Catalytic Domain; D019281:Dimerization; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D017434:Protein Structure, Tertiary; D054829:RING Finger Domains; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination
chemical_list: D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D044763:Ubiquitin-Conjugating Enzymes; C486411:Rnf4 protein, rat; D044767:Ubiquitin-Protein Ligases