title: A C2HC zinc finger is essential for the RING-E2 interaction of the ubiquitin ligase RNF125.
abstract: The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2(120-128)) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2(120-128) region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2.
authors.affiliation: Institut de Biologia Molecular de Barcelona, CSIC, Parc Cientific de Barcelona, 08028 Barcelona, Spain.
authors.forename: Marie-José
authors.initials: MJ
authors.lastname: Bijlmakers
authors.affiliation: BioNMR Laboratory, Inorganic and Organic Chemistry Department, Organic Chemistry Section, University of Barcelona, Parc Cientific de Barcelona, 08028 Barcelona, Spain.
authors.forename: João M C
authors.initials: JM
authors.lastname: Teixeira
authors.affiliation: XALOC beamline, ALBA synchrotron (CELLS), 08290 Cerdanyola del Valles, Spain.
authors.forename: Roeland
authors.initials: R
authors.lastname: Boer
authors.affiliation: Swedish NMR Centre, University of Gothenburg, SE-40530, Gothenburg, Sweden.
authors.forename: Maxim
authors.initials: M
authors.lastname: Mayzel
authors.affiliation: Institut de Biologia Molecular de Barcelona, CSIC, Parc Cientific de Barcelona, 08028 Barcelona, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Puig-Sàrries
authors.affiliation: Swedish NMR Centre, University of Gothenburg, SE-40530, Gothenburg, Sweden.
authors.forename: Göran
authors.initials: G
authors.lastname: Karlsson
authors.affiliation: Institut de Biologia Molecular de Barcelona, CSIC, Parc Cientific de Barcelona, 08028 Barcelona, Spain.|Institute for Research in Biomedicine (IRB Barcelona), Parc Cientific de Barcelona, 08028 Barcelona, Spain.
authors.forename: Miquel
authors.initials: M
authors.lastname: Coll
authors.affiliation: BioNMR Laboratory, Inorganic and Organic Chemistry Department, Organic Chemistry Section, University of Barcelona, Parc Cientific de Barcelona, 08028 Barcelona, Spain.
authors.forename: Miquel
authors.initials: M
authors.lastname: Pons
authors.affiliation: Institut de Biologia Molecular de Barcelona, CSIC, Parc Cientific de Barcelona, 08028 Barcelona, Spain.
authors.forename: Bernat
authors.initials: B
authors.lastname: Crosas
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D000072417:Protein Domains; D013329:Structure-Activity Relationship; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism*; D016335:Zinc Fingers
chemical_list: D050505:Mutant Proteins; D025801:Ubiquitin; C551868:RNF125 protein, human; D044767:Ubiquitin-Protein Ligases