Identification of PfHEUL catalytic cysteine.

A) Analysis of PfHEUL HECT domain and catalytic cysteine. AlphaFold predicted structure of PfHEUL HECT domain (residues 8211-8591) (green) overlaid with crystal structure human HUWE1 (PDB:7JQ9) (blue) and with catalytic cysteines marked by the red arrow. B) Alignment of PfHEUL and human HUWE1 HECT domains. Rendered in ESPript with catalytic cysteine demarcated by the red arrow. C) Circular Dichroism spectra of wild type (WT) and C8558A mutant PHEUL HECT recombinant protein. D) In vitro ubiquitination assay. Anti-HA western blot of an in vitro ubiquitination assay using wild type and C8558A mutant PfHEUL HECT domains to demonstrate the contribution of C8558 to the transthiolation of ubiquitin, and subsequent ubiquitination of lysine side chains.