Fig. 5: ABP mapping of the E3 cysteine nucleophile in the RZ ___domain.
From: ATP functions as a pathogen-associated molecular pattern to activate the E3 ubiquitin ligase RNF213
a Cross-linking mass spectrometry (XL-MS) analysis of the full-length RNF213-ABP complex. The residue labeled by the ABP corresponds to the active site cysteine nucleophile (yellow circle), which was identified by XL-MS. b Cross-link peptide-spectrum matches (PSMs) that have an e-value lower than 1×10-4 with a false discovery rate (FDR) of <1% are tabulated. c Representative MS2 spectrum of a crosslinked peptide corresponding to Cys4462 in murine RNF213. The spectrum is for a z = 4+ precursor ion, observed precursor mass = 2369.186 Da; theoretical crosslinked peptide mass = 2369.189 Da. d Alphafold2 model of the murine RNF213 RZ ___domain. Consistent with ABP crosslinking analysis, UV-VIS absorbance spectroscopy, and activity assays, Cys4451, His4455, Cys4471, and Cys4474 coordinate a single metal ion. Cys4462 is the active site nucleophile essential for RNF213 transthiolation activity. His4483 is suitably positioned to serve as a general base that facilitates deprotonation of substrate nucleophiles. e Autoubiquitination assay with UBE2L3 using the point mutants of cysteine and histidine residues within the RZ ___domain. Source data are provided as a Source Data file.
