Fig. 4

Specificity in HHARI/ubiquitin E2 interactions. a HHARI autoubiquitination assays. The indicated UbcH7 and UbcH5b variants were utilized in HHARI autoubiquitination assays for the indicated time points, as described in the Methods section. Time points for the timecourse experiment (represented by an extended black triangle) with UbcH7 variants were 0, 10, and 40 min. Time points for UbcH5b variants were extended to 0, 40, and 160 min to highlight the gain of function resulting from the S94K mutation relative to wild type UbcH5b. Gels were subjected to Sypro Ruby staining and α-Ub, α-UbcH7, and α-UbcH5b western blot as indicated. The asterisk indicates apparent cross-reactivity of the anti-ubiquitin antibody with UbcH7. b Intermolecular interactions at the Loop2_E3/Loop7_E2 region of the HHARI/UbcH7-Ub structure (top) and a model of HHARI in complex with UbcH5 (bottom). Zinc ions coordinated by the HHARI^RING1 domain are shown as green spheres and hydrogen bonds are indicated with black dashed lines. c Isothermal titration calorimetry data for interactions between HHARI full-length and UbcH7^WT, UbcH7^K96S, UbcH5b^WT, and UbcH5b^S94K. Upper panels show raw power data and lower panels show fits of the data to standard binding equations using NanoAnalyze software (TA instruments)