Structural insights into the mechanisms of Lys48-linked poly-ubiquitin chain synthesis by human ubiquitin-conjugating enzyme Ube2K

Abstract

Ubiquitin-conjugating enzymes (E2) form thioester bond with ubiquitin (Ub), which is subsequently transferred to the target protein for further cellular progress. Ube2K (class II E2 enzyme), a member of the human E2 family (38 members), contains a unique ubiquitin-associated (UBA) domain at its C-terminus, which is suggested to control ubiquitin recognition, dimerization, and poly-ubiquitin chain formation. Ube2K links with Ub through a thioester bond (Ube2K~Ub) and initiates Lys48-linked poly-Ub chain synthesis. I have demonstrated the changes in charging activity of Ube2K using Ube2K mutants. In addition, the structures of Ube2K linked to Ub through an isopeptide bond (Ube2K-Ub) are expected to have flexibility based on the donor Ub. Based on structural and biochemical studies, I suggest that active site gate dynamics modulate Ub conjugation activity. The closed conformation of Ube2K~Ub contributes to Lys48-linked poly-Ub chain synthesis. This enabled the confirmation of the characteristics of the initiation of poly-Ub chain synthesis through Ube2K and E3. Ube2K was also able to synthesize the Lys48-linked poly-ubiquitin chain without E3. I found that the novel interaction between acceptor di-Ub (Ub2) and auxiliary Ube2K promotes a discharging reaction and the formation of tri-Ub (Ub3), probably through the guiding and positioning of Lys48 (in the distal Ub) of the acceptor Ub2 to the active site. I also determined the crystal structure of Ube2K with Ub2 at a 2.47 Å resolution. Based on structural and biochemical data, I propose a structural model of Ub3 synthesis with Ube2K without E3.