E2-binding surface on Uba3 β-grasp domain undergoes a conformational transition.

The covalent attachment of ubiquitin (Ub) and ubiquitin-like (Ubl) proteins to various eukaryotic targets plays critical roles in regulating numerous cellular processes. E1-activating enzymes are critical, because they catalyze activation of their cognate Ub/Ubl protein and are responsible for its transfer to the correct E2-conjugating enzyme(s). The activating enzyme for neural-precursor-cell-expressed developmentally downregulated 8 (NEDD8) is a heterodimer composed of APPBP1 and Uba3 subunits. The carboxyl terminal ubiquitin-like β-grasp domain of human Uba3 (Uba3-βGD) has been suggested as a key E2-binding site defining E2 specificity. In crystal structures of free E1 and the NEDD8-E1 complex, the E2-binding surface on the domain was missing from the electron density. However, when complexed with various E2s, this missing segment adopts a kinked α-helix. Here, we demonstrate that Uba3-βGD is an independently folded domain in solution and that residues involved in E2 binding are absent from the NMR spectrum, indicating that the E2-binding surface on Uba3-βGD interconverts between multiple conformations, analogous to a similar conformational transition observed in the E2-binding surface of SUMO E1. These results suggest that access to multiple conformational substates is an important feature of the E1-E2 interaction.

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