Ubiquitin in Motion: Structural Studies of the Ubiquitin-Conjugating Enzyme∼Ubiquitin Conjugate

Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2 similar to Ub conjugate. Therefore, full characterization of the structure and dynamics of E2 similar to Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2 similar to Ub conjugates of two human enzymes, UbcH5c similar to Ub and Ubc13 similar to Ub, in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c similar to Ub conjugate populates an array of extended conformations, and the population of Ubc13 similar to Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2 similar to Ub species and thus provide insight into the diverse E2 similar to Ub protein interactome, particularly with regard to interaction with Ub ligases.