Sequence Similarity Network-Guided Discovery of a Sequence-Tolerant Cyanobactin Protease Clade for Site-Selective Peptide and Protein Modification

Abstract Proteases responsible for leader peptide removal are ubiquitous in ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic pathways. However, many characterized RiPP proteases require specific core peptide modifications prior to cleavage, limiting their broader applicability. Here, we identified a cyanobactin protease clade that efficiently processes diverse peptide substrates while maintaining strict recognition-sequence selectivity. We demonstrated that GusA, a representative member of this clade, can be used for the processing of structurally complex modified peptides and the removal of affinity tags from recombinant proteins. GusA-mediated exposure of N-terminal cysteine residues can also be applied to the construction of chemically diversified asymmetric phage-displayed macrocyclic peptide libraries, from which nanomolar-affinity ligands against Keap1 and Bcl-xL were identified, providing valuable tools for biological studies and potential therapeutics. The same strategy was further applied to site-selective and multicolor labeling of mammalian cell-surface proteins, demonstrating that GusA-mediated N-terminal cysteine generation can be readily integrated with bioorthogonal labeling chemistries. Together, our results establish cyanobactin proteases as an underexplored source of sequence-selective biocatalysts for site-selective peptide and protein modification.