Extein Residues Play an Intimate Role in the Rate-Limiting Step of Protein Trans-Splicing

[Image: see text] Split inteins play an important role in modern protein semisynthesis techniques. These naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N- and C-exteins) are strongly preferred, while other sequences cause a dramatic reduction in the splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. Here we present a thorough kinetic analysis of the relationship between C-extein composition and split intein activity. The results of these experiments were used to guide structural and molecular dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. Together, our structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity.