Extended Data Fig. 6: Generalized mechanism for CRL-dependent substrate poly-ubiquitylation.
From: Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases
a, Modelling of the RBX1-UBE2R2 ~ UBD-UBA portion of the chain formation structure (PDB code: 8PQL, this work) onto three other CRL substrate receptor complexes: SKP1-βTRCP1 (left; PDB code 6TTU), SKP1-SKP2 (middle; PDB code 7B5L), and Elongin B/C-VHL (right; PDB code 1LM8). b, Autoradiogram from quench flow, pre-steady state kinetic ubiquitylation reactions with neddylated CRL2VHL and ubiquitin-primed Hif1α peptide substrate that had been radiolabeled (indicated by *). The graph showing substrate conversion to product and the fit of the data to the model can be found in Extended Data Fig. 7a. The autoradiogram is representative of triplicate technical replicates. UB, ubiquitin. c, Graph corresponding to ubiquitylation reactions containing neddylated CRL1FBXW7 with wild-type (WT) or mutant ubiquitin-primed cyclin E peptide substrates and WT or mutant UBE2R2. The data were fit to closed form solutions using Mathematica19 (v.13.1). Datapoints from triplicate technical replicates are shown. d, Superposition of the CUL2 C-terminal domains from the CRL2VHL-MZ1-BRD4-UBA-UBE2R2~UBD (gray) and CRL2FEM1C-Sil1- UBA-UBE2R2~UBD structures (green and blue), showing translation of the RBX1 RING domains that enables repositioning of UBE2R2~ubiquitin relative to substrate. e, Autoradiogram for BRD4BD2 ubiquitylation reactions in the presence of neddylated CRL2VHL, the PROTAC MZ1, UBE2R2 and WT ubiquitin. Notice the sequential appearance of ubiquitin-primed BRD4 (BRD4BD2-UB) and poly-ubiquitylated BRD4 (BRD4BD2-UB-UB) which enabled estimation of the rate of ubiquitin transfer to primed neo-substrate (Fig. 4e and Extended Data Table 1; see methods). The autoradiogram is representative of triplicate technical replicates. f, Graphs showing depletion of BRD4BD2 (left) or the appearance of primed neo-substrate (right) in the presence of the indicated UBE2R2 proteins and the fit of the data to the model. UBA, acceptor ubiquitin; UBD, donor ubiquitin; BRD4BD2, BRD4 (residues 346-460); ELOB, Elongin B; ELOC, Elongin C.
