Fig. 1: Nucleotide-dependent regulation of RNF213 E3 activity.
From: ATP functions as a pathogen-associated molecular pattern to activate the E3 ubiquitin ligase RNF213
a Schematic of RNF213 sub-modules (functional AAA3 and AAA4 in green) and mutations used to probe the role of the two functional AAA units in E3 ligase activity (WA3, K2387A; WA4, K2736A; WB3, E2449Q; WB4, E2806Q; WB34, E2449Q E2806Q; murine numbering). b Representative progress curves for the fluorescence polarization-based E3 activity assay. Reactions were carried out with wild type or mutant RNF213 in the presence of ubiquitin labeled sub-stoichiometrically with DyLight488. The time interval (30-60 min) used to determine rate constants is depicted in gray. c Rates of ubiquitin adduct formation (relative to wild type protein). d Discharge of Ub from purified UBE2L3~Ub discharge assay in the presence of RNF213 and various nucleotides. e Quantification of the discharge extent (free E2 / total E2). f Time course of UBE2L3~Ub discharge assay, using different nucleotide concentrations. g Schematic of the nucleotide-dependent activation of RNF213 HECT-like E3 activity, required to form a covalent complex with an activity-based probe (ABP, wavy bond corresponds to a triazole-ethyl linker40). h Recombinant RNF213 was tested for transthiolation activity with a biotin-tagged ABP, in the presence or absence of 1 mM nucleotides indicated. Samples were resolved by SDS-PAGE and visualized by two-channel near-IR immunoblot using anti-RNF213 antibody (blue) and streptavidin (white). Panel representative of n = 3. i ABP analysis of ATPγS-dependent RNF213 activation in the presence of either ADP (900 μM) or AMP (900 μM). j Quantification of biotin signal determined in i. Mean values are plotted and non-linear fit curves shown (n = 2). Source data are provided as a Source Data file.
