Extended Data Fig. 3: SAH-UBE2A binds to UBE1 in the presence and absence of ubiquitin and ATP.
From: Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer
a, Similar protection of SAH-UBE2A from deuterium exchange upon incubation with UBE1 (2:1 protein:peptide) in the presence (black) or absence (light gray) of ubiquitin and ATP. The relative difference in deuterium uptake after 10 s of labeling is shown. The difference in uptake was calculated from the mean deuterium level for SAH-UBE2A in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. b, C-terminally FITCylated SAH-UBE2A bound to UBE1 with respective Kds of 384 ± 43 nM and 359 ± 60 nM in the absence (light gray) and presence (black) of ubiquitin and ATP, as assessed by fluorescence polarization assay. Kds were determined by nonlinear regression analysis of experiments performed in technical triplicate (with ubiquitin/ATP) and plotted alongside the data shown in Fig. 2i (without ubiquitin/ATP). The two curve fits have R2 values of 0.9491 (without ubiquitin/ATP) and 0.9636 (with ubiquitin/ATP). Source data for the HXMS and FP plots are available online.
