Supplementary Figure 1: Definition and assessment of cIAP1 constructs.

(a) cIAP1 constructs used in this study are shown as primary structure schematics with domains colored as in the main text. Mutations and deletions are indicated with magenta lines. The mutations in the cIAP1-B3R construct reduce proteolysis and non-native disulfide formation¹. (b) The binding of cIAP1-B3R (blue) and cIAP1-B3R_MF-AA (gray) to monobiotinylated ubiquitin was tested using biolayer interferometry. Equilibrium response values are plotted against cIAP1 concentration. No detectable binding is observed by the cIAP1-B3R_MF-AA construct. cIAP1-B3R binds ubiquitin with a K_D of approximately 20 μM as determined by a single-site binding isotherm fit, in good agreement with past studies². Recent reports have suggested that this mutation destabilizes cIAP1³, which might contribute to the very slight decrease in E2 binding affinity we observe in the MF-AA variants. The use of cIAP1_MF-AA in E2 binding studies removes the complicating effects of direct cIAP1-ubiquitin binding in the case of the E2-Ub conjugate. (c) The binding of cIAP1-B3R (blue) and cIAP1-B3R-ΔCARD (green) to E2_SRCK was assessed using biolayer interferometry. Equilibrium response values were plotted as a function of E2 concentration, and the resulting curves were fit to a single-site binding isotherm. The affinities determined are approximately 27 μM (cIAP1-B3R) and 19 μM (cIAP1-B3R-ΔCARD), recapitulating the relative increase in affinity upon CARD deletion observed in the case of cIAP1-B3R_MF-AA.