Non‐canonical inteins: Protein splicing by alternate mechanisms

Abstract

Protein splicing is the post‐translational excision of an intervening polypeptide (intein) from its flanking domains (the exteins), concomitant with extein ligation. The first step of splicing is an amide‐to‐thioester rearrangement of the peptide bond linking the N‐extein and intein. Class three inteins bypass this step. The Clostridium thermocellum TerA intein is class three, whereas the Thermobifida fusca Tfu2914 intein is a traditional intein despite having class three sequence motifs. The third step of splicing is Asn cyclization coupled to cleavage of the peptide bond linking the intein and C‐extein. Both the Methanoculleus marisnigri (Mma) and the Pyrococcus abyssi (Pab) PolII inteins promote efficient splicing with C‐terminal Gln in place of Asn. The activity of the Mma PolII intein may be regulated by disulfide bond formation. The Pab PolII intein splices only at elevated temperatures, allowing for isolation of a stable precursor and study each step of splicing in vitro. An NMR solution structure of the Pab PolII intein reveals a particularly rigid structure, a disordered loop absent in a highly similar P. horikoshii intein, and a β‐hairpin specific to thermophilic inteins.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and the Camille and Henry Dreyfus Foundation.