Conformational Dynamics Modulate Activation of the Ubiquitin Conjugating Enzyme Ube2g2.

The ubiquitin conjugating enzyme Ube2g2 together with its cognate E3 ligase gp78 catalyzes the synthesis of lysine-48 polyubiquitin chains constituting signals for the proteasomal degradation of misfolded proteins in the endoplasmic reticulum. Here, we employ NMR spectroscopy in combination with single-turnover diubiquitin formation assays to examine the role of the RING domain from gp78 in the catalytic activation of Ube2g2∼Ub conjugates. We find that approximately 60% of the Ube2g2∼Ub conjugates occupy a closed conformation in the absence of gp78-RING, with the population increasing to 82% upon gp78-RING binding. As expected, strong mutations in the hydrophobic patch residues of the ∼Ub moiety result in Ube2g2∼Ub populating only open states with corresponding loss of the ubiquitin conjugation activity. Less disruptive mutations introduced into the hydrophobic patch of the ∼Ub moiety also destabilize the closed conformational state, yet the corresponding effect on the ubiquitin conjugation activity ranges from complete loss to an enhancement of the catalytic activity. These results present a picture in which Ube2g2's active site is in a state of continual dynamic flux with the organization of the active site into a catalytically viable conformation constituting the rate-limiting step for a single ubiquitin ligation event. Ube2g2's function as a highly specific K48-polyubiquitin chain elongator leads us to speculate that this may be a strategy by which Ube2g2 reduces the probability of nonproductive catalytic outcomes in the absence of available substrate.