Extended Data Fig. 1: Formation of an activity-based probe to capture millisecond poly-ubiquitin chain formation.
From: Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases
a, Coomassie-stained SDS-PAGE gel examining reactions containing wild-type (WT) or catalytically inactive UBE2R2 with the ligation mimic (defined as the donor ubiquitin cross-linked to the BmDPA molecule, see Methods) and in the absence or presence of neddylated CRL2FEM1C or separated CRL components. The red box identifies the “trapped” UBE2R2 ~ UBD-substrate-UBA product. Notice that formation of the trapped complex was dependent on the presence of WT UBE2R2 containing its C-terminal acidic tail and an intact CRL containing the substrate receptor complex (FEM1C-ELOBC, Elongin B/C-FEM1C; UBD, donor ubiquitin; UBA, acceptor ubiquitin; UBE2R2 FL, full-length; MW, molecular weight). The gel is representative of duplicate technical replicates. b, Electron density from the composite cryo-EM map highlighting the UBE2R2 active site region including UBD’s C-terminal tail, UBA, and the UBE2R2 synergy loop. While visualization of UBA’s Lys48 was not possible owing to its replacement with a Cys to promote trap formation, clear density is visible for synergy loop residue His98, previously shown to be important for UBE2R2 activity32, whose side-chain imidazole ring points towards the UBE2R2 active site Cys93. c, Select regions of the composite cryo-EM map and ribbon diagrams of the corresponding protein subunits. Ball-and-sticks are shown for residues where side-chains were built. Top-left, the central stalk of CUL2; top-right, donor ubiquitin (UBD) and RBX1; middle-left, the “trap” indicating the location for the ligation mimic-UBE2R2-crosslinked junction; middle-right, Elongin B/C with part of FEM1C; bottom, the catalytic core with UBE2R2, RBX1, and donor and acceptor ubiquitins (UBD and UBA, respectively).
