Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein

Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled to ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.

Research highlights
► The Pyrococcus abyssi lon protease intein promotes efficient protein splicing.
► Inteins with mutations that interfere with individual steps of splicing do not promote unproductive side reactions.
► The intein splices with Lys in place of the highly conserved penultimate His.
► The intein is flanked by a Gly-rich region at its C terminus that may increase the efficiency of the third step of splicing, Asn cyclization coupled to peptide bond cleavage.