Abstract
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.
Highlights
Culminating in the attachment to substrate of polyubiquitin chains linked together by isopeptide bonds formed between the C terminus of one ubiquitin and the Lys-48 side chain of another ubiquitin [2, 3]
Ϫ3.3 Ϫ5.7 Ϫ9.3 a Number of models clustered within 2 Å r.m.s.d. among the 10 lowest scoring solutions. b paramagnetic relaxation enhancement (PRE) violations greater than 15 Å. c The intermolecular component of the total energy of the complex expressed in Rosetta energy units, where negative indicates stronger binding. d The PREs utilized for the primary solution were: Ub-K48C-MTSL % Ube2g2-(74, 75); Ub-G75CD-MTSL % Ube2g2-(23, 45, 50 –52, 156, 159, 161–165). e The PREs utilized for the alternate solution were: Ub-K48C-MTSL % Ube2g2-23; Ub-G75C⌬-MTSL % Ube2g2-(4, 5, 11–15, 17, 18, 26, 27, 30, 33, 34, 38). f The proximal subunit clustered within 5 Å r.m.s.d
Ube2g2 binds to the hydrophobic patch of ubiquitin with an interface composed of residues in its -sheet and C-terminal helix, which is consistent with reported observations for other E2 apoenzymes including HsUbc2b [19] and UbcH5c [20]
Summary
BRCA1-mediated ubiquitin chain elongation in vitro [20]. These results suggest the involvement of multiple E2 proteins as well as raising the question as to whether E2 proteins might interact with polyubiquitin chains differently than with ubiquitin alone. We find that Ube2g2 exhibits a clear preference for binding to the distal subunit of the Lys48-linked diubiquitin molecule (containing the free Lys-48 side chain) presumably due to steric hindrance of proximal subunit binding by the Lys-48 —Gly-76 isopeptide linkage connecting the two ubiquitin molecules. These results suggest that Ube2g2 will in general preferentially bind to the terminal ubiquitin subunit of a Lys-48-linked polyubiquitin chain
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