Background:The PolII intein from the hyperthermophilic Pyrococcus abysii only splices at very high temperature. Results: NMR structure, dynamics, and mutagenesis of Pab PolII intein have been characterized. Conclusion:The Pab PolII intein has unique structural and dynamic features that may contribute to its higher temperature for optimal activity. Significance: Pab PolII intein is an ideal candidate for protein engineering.
Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.
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.
Inteins are intervening polypeptides that are excised during the process of protein splicing. Protein splicing is a self‐catalyzed process in which the intein is removed and the flanking polypeptides are ligated (N‐ and C‐ exteins). The Pyrococcus abyssi (Pab) PolII intein only splices at elevated temperatures. An NMR structure of the intein recently was solved in a collaborator's lab (Du, Z., et al. (2011) Structural and Mutational Studies of a Hyperthermophilic Intein from DNA Polymerase II of Pyrococcus abyssi. J. Biol. Chem. 286(44) 38638–38648.). The NMR data suggest increased rigidity of the intein in comparison to a mesophilic intein from Mycobacterium tuberculosis (Mtu), as well as a β‐hairpin found only in the structures of thermophilic inteins. We have replaced the β‐hairpin from the Pab intein with a linker inspired by the Mtu intein sequence to determine if the β‐hairpin plays a role in the structural stability/rigidity of the intein. We also have designed a genetic screening system that links protein splicing to cell growth on kanamycin in order to select for mutants that allow for splicing at lower temperatures.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KVM), and the Arnold and Mae Beckman Foundation (KMC).
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