The mechanism of intein-mediated protein splicing
Protein splicing is so rapid that the precursor protein is rarely observed in native systems. The intein plus the first C-extein residue contain sufficient information for splicing in foreign proteins. However, exteins may affect splicing rates or efficiency. Splicing in foreign protein contexts often results in an increase in dead-end cleavage reaction products.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.
A. The protein splicing mechanism depicted with Ser at both splice junctions
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.
B. 
Animation of The Protein Splicing Pathway
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C. An Alternative Protein Splicing Mechanism for Inteins that Naturally Begin with Ala.
Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. Several families of inteins have been identified that begin with Ala rather than the consensus nucleophiles, Ser or Cys. In standard inteins, an N-terminal Ser, Cys or Thr is absolutely required for splicing. An N-terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N-terminal splice junction (thio)ester. However, experiments with the M. jannaschii KlbA intein demonstrated that Ala1 inteins can splice efficiently using an alternative protein splicing mechanism (Southworth 2000). In this non-canonical pathway, the C-extein nucleophile (Ser, Cys or Thr) attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is identical: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins. Just like standard inteins, the Mja KlbA intein also requires the help of the conserved Thr and His in Block B to activate the N-terminal splice junction. We have also demonstrated splicing of the Mle DnaB intein (dnaB-b insertion site, E. Davis, M. Southworth & F. Perler, unpublished data) which is another Ala1 intein, suggesting that different families of naturally occurring Ala1 inteins should be capable of splicing.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.
D. Similarities between inteins and hedgehog protein autoprocessing domains
Hedgehog proteins are signaling molecules required for embryonic pattern formation (Beachy 1997). They are synthesized as inactive precursors with an N-terminal signaling domain linked to a C-terminal autoprocessing domain (Hh-C). Hh-C begins with a Cys that undergoes an acyl rearrangement analogous to Step 1 of the protein splicing pathway. Hh-C also has sequence similarity to inteins with conserved sequences corresponding to intein Blocks A and B (Koonin 1995). In a transesterification reaction similar to Step 2 of the protein splicing pathway, the hydroxyl group of cholesterol attacks this thioester bond, resulting in attachment of cholesterol to the C-terminus of the hedgehog protein signaling domain. Cholesterol anchors the signaling domain to the cell surface. The Drosophila Hh-C domain is composed of a subdomain that directs thioester formation, followed by a sterol recognition region required for cholesterol transfer. Several nematode Hh-C domains contain unrelated C-terminal extensions that may interact with molecules other than cholesterol and have been tentatively termed Adduct Recognition Regions (Beachy 1997). Crystal structure analysis (see below) indicates that Hedgehog autoprocessing domains evolved from a common ancestor and that higher organisms redirected the ability of inteins to ligate flanking peptides and utilized these modified inteins to ligate lipids to the hedgehog signaling domain for compartmentalization at the cell surface (which is required for signaling).
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E. Intein Structure
Not only is there sequence and mechanistic similarities between inteins
and hedgehog protein autoprocessing domains, but there is a high degree
of structural identity amongst main chain alpha carbon atoms. This structural
similarity led Leahy and coworkers to propose that inteins and Hh-C have
a common structural fold (Hall
1997). This conserved structure is called a Hint
module (Hedgehog, INTein). The main chain alpha carbon
atoms of 100 amino acids in the Mxe GyrA intein are superimposable onto
the Hint module fold despite the fact that there is little amino acid sequence
identity (Klabunde
1998). Furthermore, Leahy and coworkers have proposed that inteins
and Hh-C evolved from a common precursor (Hall
1997 and Beachy
1997).
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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Return to Top - Hodges 1992
- Xu 1993
- Xu 1994
- Shao 1995
- Xu 1996
- Chong 1996
- Shao 1996
- Duan 1997
- Hall 1997
- Heath 1997
- Kawasaki 1997
- Nogami 1997
- Wang 1997
- Shao 1997B
- Anraku 1997B
- Derbyshire 1997
- Klabunde 1998
- Perler 1998
- Paulus1998B
- Chong 1998
- Wood 1999
- Chen 2000
- Noren 2000
- Paulus 2000
- Poland 2000
- Southworth 2000
F. Selected Mechanism References:
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Last database update: 11/05/10