Ubiquitin-conjugating enzymes (E2) form thioester bonds with ubiquitin (Ub), which are subsequently transferred to target proteins for cellular progress. Ube2K/E2-25K (a class II E2 enzyme) contains a C-terminal ubiquitin-associated (UBA) domain that has been suggested to control ubiquitin recognition, dimerization, or poly-ubiquitin chain formation. Ube2K is a special E2 because it synthesizes K48-linked poly-ubiquitin chains without E3 ubiquitin ligase. We found that a novel interaction between the acceptor di-Ub (Ub(2)) and the auxiliary Ube2K promotes the discharging reaction and production of tri-Ub (Ub(3)), probably by guiding and positioning the K48 (in the distal Ub) of the acceptor Ub2 in the active site. We also determined the crystal structure of Ube2K-Ub(2) at 2.47 angstrom resolution. Based on our structural and biochemical data, we proposed a structural model of Ub3 synthesis by Ube2K without E3. (C) 2018 Elsevier Inc. All rights reserved.