Non-canonical residues of the marginally stable monomeric ubiquitin conjugase from goldfish are involved in binding to the C terminus of Ring 1B

E2 ubiquitin conjugases are ~ 20 kDa 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-Cca 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-Cca towards non-canonical E3 ligases. Binding of E2-Cca 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 ~ 400 nM, as shown by fluorescence and ITC. Furthermore, binding of E2-Cca 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-Cca comprises the first β-strand and nearby residues.

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► The conformational stability of E2-Cca is very low.
► The C-terminal region of Ring1B, C-Ring1B, binds E2-Cca, with a 400 nM affinity.
► The E2-Cca-binding region of C-Ring1B does not involve any RING finger.
► The C-Ring1B-binding region of E2-Cca does not involve the canonical loops.
► Residues M43 and F53 of E2-Cca are critical in the binding of both proteins.