Recruitment of Ubiquitin within an E2 Chain Elongation Complex.

Recruitment of Ubiquitin within an E2 Chain Elongation Complex.
Biophys J. 2020 Feb 15;:
Authors: Cook BW, Lacoursiere RE, Shaw GS
Abstract
The ubiquitin (Ub) proteolysis pathway uses an E1, E2, and E3 enzyme cascade to label substrate proteins with ubiquitin and target them for degradation. The mechanisms of ubiquitin chain formation remain unclear and include a sequential addition model, in which polyubiquitin chains are built unit by unit on the substrate, or a preassembly model, in which polyubiquitin chains are preformed on the E2 or E3 enzyme and then transferred in one step to the substrate. The E2 conjugating enzyme UBE2K has a 150-residue catalytic core domain and a C-terminal ubiquitin-associated (UBA) domain. Polyubiquitin chains anchored to the catalytic cysteine and free in solution are formed by UBE2K supporting a preassembly model. To study how UBE2K might assemble polyubiquitin chains, we synthesized UBE2K-Ub and UBE2K-Ub2 covalent complexes and analyzed E2 interactions with the covalently attached Ub and Ub2 moieties using NMR spectroscopy. The UBE2K-Ub complex exists in multiple conformations, including the catalytically competent closed state independent of the UBA domain. In contrast, the UBE2K-Ub2 complex takes on a more extended conformation directed by interactions between the classic I44 hydrophobic face of the distal Ub and the conserved MGF hydrophobic patch of the UBA domain. Our results indicate there are ...

Source: Biophysical Journal - February 14, 2020 Category: Physics Authors: Cook BW, Lacoursiere RE, Shaw GS Tags: Biophys J Source Type: research