This section describes the mechanism of intein-mediated protein splicing, including:
Protein splicing involves 4 nucleophilic displacements by the 3 conserved splice junction residues. Acids and bases or hydrogen bonding residues that assist these nucleophilic displacements are omitted in the figure below. The intein penultimate His in Block G assists in Asn cyclization and C-terminal cleavage (Xu 1996) by hydrogen bonding to the Asn carbonyl oxygen, making this peptide bond more labile (Klabunde 1998, Duan 1997). The Thr and His in Block B assist in the initial acyl rearrangement at the N-terminal splice junction (Kawasaki 1997) by hydrogen bonding to main chain atoms and holding the residue preceding the intein in a non-standard cis conformation (Klabunde 1998) or in a strained conformation (Poland 2000). Any residue that can form similar hydrogen bonds can substitute for these conserved facilitating residues in Blocks B and G. The mechanism of protein splicing has recently been reviewed in Noren 2000, Paulus 2000, Perler 1997C, Shao 1997 and Perler 1998. Several previous reviews contain mechanisms now known to be incorrect.
STEP 1: The N-terminal splice junction is activated by a N-O or N-S acyl rearrangement at the intein N-terminus that moves the N-extein to the side chain of the Ser/Cys at the intein N-terminus, forming the linear ester/thioester intermediate. A few inteins have been identified with a N-terminal Ala (A) (see Splicing motifs), although splicing has not been demonstrated with these inteins. Ala cannot undergo an acyl shift like Ser/Thr/Cys, since it doesn't have an hydroxyl/thiol side chain. However, these inteins may be active if the residues facilitating the reaction are still making the splice junction peptide bond more labile and if the C-extein Ser/Thr/Cys is in the proper position to attack the splice site; in this case, the downstream splice junction Ser/Thr/Cys would directly cleave the N-terminal splice junction peptide bond (see splicing pathway A in Xu 1994) to form the branch intermediate.
STEP 2: The upstream ester/thioester bond is attacked during a transesterification reaction by the hydroxyl/thiol group of the C-extein Ser/Thr/Cys, resulting in cleavage at the N-terminal splice junction and transfer of the N-extein to the side chain of the C-extein Ser/Thr/Cys, forming the branched protein intermediate.
STEP 3: The branch is resolved by cyclization of the conserved intein C-terminal Asn to form a succinimide ring, resulting in cleavage of the C-terminal splice junction. The succinimide can be hydrolyzed to form Asn or isoasparagine. A few inteins have been identified with a C-terminal Gln (Q) (see Splicing motifs); although splicing has not been demonstrated with these inteins, Gln is capable of undergoing a cyclization reaction just like Asn and should thus be able to substitute for Asn.
STEP 4: A spontaneous 0-N or S-N acyl rearrangement results in formation of a native peptide bond between the exteins.
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1. See the animation of The Protein Splicing Pathway with FLASH or QuickTime.
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The KlbA and Mle DnaB inteins have overcome the barriers to direct nucleophilic attack on the peptide bond at the N-terminal splice junction that are present in previously studied inteins with Ser or Cys at their N-terminus. It is unclear why other inteins can't perform similar reactions, since the Block B oxyanion hole is still available to facilitate direct attack on the N-terminal splice junction. Possibly, (thio)ester formation may be necessary in standard inteins to align the C-extein nucleophile, to remove steric hindrances or to induce a conformational shift that allows attack by the +1 nucleophile (Cys, Ser or Thr). The crystal structure of a S.cerevisiae VMA intein precursor has helped to resolve this question by revealing that Cys+1 is too far away to directly attack either a peptide or a thioester bond at the N-terminal splice junction, leading the authors to suggest that inteins must undergo a conformational shift to allow attack by the Cys+1 nucleophile (Poland 2000). We propose that Cys+1 (or its equivalent residue) in Ala1 inteins is already in position to attack the N-terminal splice junction amide bond in the precursors protein.
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Hint modules are composed of ~12 beta-strands. In inteins, the core endonuclease or linker region is inserted into the Hint module between intein Blocks N4 and F. The Sce VMA intein has an additional endonuclease DNA recognition region (DRR) between Blocks B and N4 (Duan 1997, Hall 1997 and Perler 1998) that is not present at this position in most other inteins. The core endonuclease domain is composed of both beta-strands and alpha-helices. The structure of the Sce VMA intein core endonuclease domain (Duan 1997) is very similar to the structure of a dimer of the intron encoded endonuclease, I-CreI (Heath 1997). Both PI-SceI and I-CreI are members of the LAGLIDADG (DOD) family of homing endonucleases.
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