Extended Data Figure 4. SUMO^B Bound to the E2 Backside enhances E2_Ubc9-SUMO Recruitment.

a, Alignment of the current E2_Ubc9/backside SUMO^B (left) to previously observed E2_Ubc9/backside SUMO complexes (right). The position of the D68R mutation is shown in red sticks (left). b, Primary E3_Siz1 structure (top). Cartoons indicating the E3_Siz1 or E3_Siz1-SUMO fusion constructs utilized in the multiple turnover in vitro assays (middle) shown in Fig. 3 utilizing a titration of the purified E2_Ubc9-SUMO^D68R-Alexa488 thioester with or without 1.5-fold excess of the indicated additional molecule of non-conjugatable SUMO, 1 nM of the indicated E3 construct and 32 µM PCNA. Representative non-reducing SDS-PAGE showing the 0.5 µM E2_Ubc9-SUMO^D68R-Alexa488 thioester reactions below the plots of the rates of reaction for each E2_Ubc9-SUMO^D68R concentration (middle). The kinetics of SUMO modification of PCNA were calculated and the K_m and k_cat were determined (bottom) and are also shown in Extended Data Table 2. The quantified rate data show mean ± s.d. (n=3 technical replicates). For gel source data, see Supplementary Figure 1.