Extended Data Fig. 4: Unanchored di-ubiquitin chain synthesis enables estimation of the Km and the apparent affinity of acceptor ubiquitin for UBE2R-family E2s.
From: Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases
a, Autoradiogram showing UBD-UBA formation for reactions containing UBE2R2, neddylated CUL2-RBX1, and increasing UBA concentrations. The gel migrations of radiolabeled UBD (indicated by *) and UBA-UBD product are shown (top). The graph shows the fraction of UBA-UBD product formed as a function of the unanchored UBA concentration with wild-type (WT) components (bottom). The data were fit to the Michaelis-Menten model (GraphPad Prism software v9) to estimate the Km of UBA for the UBE2R2-mediated poly-ubiquitylation complex. The autoradiogram is representative of triplicate technical replicates. UBD, donor ubiquitin; UBA, acceptor ubiquitin. b, Graphs showing the fraction of di-ubiquitin product formation (UBD-UBA) as a function of the unanchored acceptor ubiquitin (UBA) concentration with WT or mutant protein components (the assay setup is shown in Extended Data Fig. 3e). The data were fit to the Michaelis-Menten model using non-linear regression (GraphPad Prism software v9). Datapoints from triplicate technical replicates are shown. c, Autoradiograms showing time courses for the formation of free UBA-UBD product with WT UBE2R2. Reactions were performed in the absence (w/o) or presence of WT neddylated CUL2-RBX1 or a mutant harboring a R91E RBX1 subunit. All autoradiograms are representative of triplicate technical replicates. d, Graph showing product formation normalized to the ratio of the UBD and UBE2R2 concentrations with respect to time. Rates were derived by linear regression in Prism. Estimates are based on n = 3 technical replicates. e, Coomassie-stained SDS-PAGE gel comparing the loading of WT UBE2R2 with K48R or K48R/R74E donor ubiquitins and upon titration of E1 enzyme (top). The graph shows the fraction of the total ubiquitin thioesterified to UBE2R2 (bottom; based on n = 2 technical replicates). The results determined the E1 concentration in subsequent experiments with K48R/R74E donor ubiquitin (see methods). The gel is representative of duplicate technical replicates. UB, ubiquitin. f, Bar graphs comparing the Km values of unanchored UBA for UBE2R1 in the presence of neddylated CUL2-RBX1 and the indicated mutants. Bars showing a ‘>‘ reflect reactions where saturation of UBE2R2 with UBA was not feasible (the top concentration in the dilution series is shown). The value of each bar represents the estimated value for Km based on n = 3 technical replicates.
