title: Non-canonical residues of the marginally stable monomeric ubiquitin conjugase from goldfish are involved in binding to the C terminus of Ring 1B.
abstract: E2 ubiquitin conjugases are ~20kDa enzymes involved in ubiquitination processes in eukaryotes. The E2s are responsible for the transference of ubiquitin (Ub) to E3 enzymes, which finally transfer Ub to diverse target proteins, labelling them for degradation, localization and regulation. Although their functions are relatively well-characterized, their conformational stabilities are poorly known. In this work, we have used, as a model for our biophysical and binding studies, the E2-C from Carassius auratus (goldfish), a homologue of the human ubiquitin conjugase UbcH10. E2-C(ca) was a monomeric protein with an elongated shape; moreover, the protein was only marginally stable within a narrow pH range (from 6.0 to 8.0). We also explored the binding of E2-C(ca) towards non-canonical E3 ligases. Binding of E2-C(ca) to the C terminus of murine Ring 1B (C-Ring1B), which does not contain the RING finger of the whole Ring1B, occurred with an affinity of ~400nM, as shown by fluorescence and ITC. Furthermore, binding of E2-C(ca) to C-Ring1B did not occur at its canonical E2-loops, since residues M43 and F53, far away from those loops, were involved in binding. Thus, the C-Ring1B-interacting region of E2-C(ca) comprises the first β-strand and nearby residues.
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Alicante, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Aguado-Llera
authors.forename: Rosa
authors.initials: R
authors.lastname: Doménech
authors.forename: Marco
authors.initials: M
authors.lastname: Marenchino
authors.forename: Miguel
authors.initials: M
authors.lastname: Vidal
authors.forename: José L
authors.initials: JL
authors.lastname: Neira
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D029941:Fish Proteins / Q000737:chemistry*; D006054:Goldfish; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D055550:Protein Stability; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029941:Fish Proteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases