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.

Mechanism of polyubiquitin chain recognition by the human ubiquitin conjugating enzyme Ube2g2.

Ube2g2 is a human ubiquitin conjugating (E2) enzyme involved in the endoplasmic reticulum-associated degradation pathway, which is responsible for the identification and degradation of unfolded and misfolded proteins in the endoplasmic reticulum compartment. The Ube2g2-specific role is the assembly of Lys-48-linked polyubiquitin chains, which constitutes a signal for proteasomal degradation when attached to a substrate protein. NMR chemical shift perturbation and paramagnetic relaxation enhancement approaches were employed to characterize the binding interaction between Ube2g2 and ubiquitin, Lys-48-linked diubiquitin, and Lys-63-linked diubiquitin. Results demonstrate that ubiquitin binds to Ube2g2 with an affinity of 90 µM in two different orientations that are rotated by 180° in models generated by the RosettaDock modeling suite. The binding of Ube2g2 to Lys-48- and Lys-63-linked diubiquitin is primarily driven by interactions with individual ubiquitin subunits, with a clear preference for the subunit containing the free Lys-48 or Lys-63 side chain (i.e. the distal subunit). This preference is particularly striking in the case of Lys-48-linked diubiquitin, which exhibits an ∼3-fold difference in affinities between the two ubiquitin subunits. This difference can be attributed to the partial steric occlusion of the subunit whose Lys-48 side chain is involved in the isopeptide linkage. As such, these results suggest that Lys-48-linked polyubiquitin chains may be designed to bind certain proteins like Ube2g2 such that the terminal ubiquitin subunit carrying the reactive Lys-48 side chain can be positioned properly for chain elongation regardless of chain length.

Expression and purification of aspartate beta-semialdehyde dehydrogenase from infectious microorganisms.

l-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) lies at the first branch point in the aspartate metabolic pathway that leads to the formation of the amino acids lysine, isoleucine, methionine, and threonine in most plants, bacteria, and fungi. Since the aspartate pathway is not found in humans, but is necessary for bacterial cell wall biosynthesis, the enzymes in this pathway are potential targets for the development of new antibiotics. The asd gene that encodes for ASA DH has been obtained from several infectious organisms and ligated into a pET expression vector. ASA DHs from Haemophilus influenza, Pseudomonas aeruginosa, and Vibrio cholerae were expressed as soluble proteins in Escherichia coli, while ASA DH from Helicobacter pylori was obtained primarily as inclusion bodies. The V. cholerae genome contains two asd genes. Both enzymes have been expressed and purified, and each displays significant ASA DH activity. The purification of highly active ASA DH from each of these organisms has been achieved for the first time, in greater than 95% purity and high overall yield. Kinetic parameters have been determined for each purified enzyme, and the values have been compared to those of E. coli ASA DH.