Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail

Background: Ubiquitin-conjugating enzymes (E2s) are central enzymes involve d in ubiquitin-mediated protein degradation. During this process, ubiquitin (Ub) and the E2 protein form an unstable E2-Ub thiolester intermediate pri or to the transfer of ubiquitin to an E3-ligase protein and the labeling of a substrate for degradation. A series of complex interactions occur among the target substrate, ubiquitin, E2, and E3 in order to efficiently facilit ate the transfer of the ubiquitin molecule. However, due to the inherent in stability of the E2-Ub thiolester, the structural details of this complex i ntermediate are not known. Results: A three-dimensional model of the E2-Ub thiolester intermediate has been determined for the catalytic domain of the E2 protein Ubcl (Ubc1(Delt a 450)) and ubiquitin from S. cerevisiae. The interface of the E2-Ub interm ediate was determined by kinetically monitoring thiolester formation by H-1 -N-15 HSQC spectra by using combinations of N-15-labeled and unlabeled Ubc1 (Delta 450) and Ub proteins. By using the surface interface as a guide and the X-ray structures of Ub and the 1.9 Angstrom structure of Ubc1(Delta 450 ) determined here, docking simulations followed by energy minimization were used to produce the first model of a E2-Ub thiolester intermediate. Conclusions: Complementary surfaces were found on the E2 and Ub proteins wh ereby the C terminus of Ub wraps around the E2 protein terminating in the t hiolester between C88 (Ubc1(Delta 450)) and G76 (Ub). The model supports in vivo and in vitro experiments of E2 derivatives carrying surface residue s ubstitutions. Furthermore, the model provides insights into the arrangement of Ub, E2, and E3 within a ternary targeting complex.