Fig. 2: Characterization of TseI cleavage and key residues.

a Cleavage sites determined by N-terminal sequencing. Each band was excised for N-terminal Edman sequencing as well as LC-MS/MS identification (see also Supplementary Fig. 3A). b Weblogo depicting conserved residues of Rhs N-/C-terminal sequences deriving from sequence alignment of 48 representative Rhs homologs. Sequences are provided in Supplementary Data 1. Black arrows indicate the predicted key activity residues that are mutated in this study while gray arrows indicate the first residue of Rhs and VIRC post cleavage, respectively. c Western blotting analysis of TseI and its cleavage-defective mutants. All constructs were cloned to pETDUET1 vectors with an N-terminal FLAG tag and a C-terminal 3V5 tag. Proteins were induced in E. coli with 0.01 mM IPTG. The nontoxic HFH-AAA TseI mutant is used as the parental construct. The same pETDUET1 constructs were also used for in vitro expression shown in d. In vitro expression was performed with a PURExpress^® In Vitro Protein Synthesis Kit following the manufacturer's instruction. Synthesized proteins were subject to SDS-PAGE analysis, followed by western blot analysis with anti-FLAG and anti-V5 antisera. Source data are provided as a Source Data file. Data in a, c, d are representative of at least two replications.