Protein Splicing

Protein splicing is the excision of an intervening protein sequence (the INTEIN) from a protein precursor and then the concomitant ligation of the remaining protein fragments (the EXTEINS) to an intact, mature extein host protein. (Perler et al., 1994) The splicing leads to a native peptide bond between the ligated exteins (Cooper et al., 1993). The splicing reaction is self-catalytic and requires no external energy sources or cofactors (Tori et al., 2010).  

The fundamental path of protein splicing

The fundamental path of protein splicing

IN­TEINS

Inteins are the protein equivalent of introns, and they invade host genes via homing endonuclease (HEN) domains. During post-translational maturation of a protein, inteins excise themselves from their host protein (extein) and ligate the two remaining surrounding sequences. Therefore, the splicing activity of inteins are necessary for the host’s correct maturation and function. (Tori and Perler, 2011)  

Research has split inteins into three classes: class 1, class 2 and most recently discovered class 3. The classification is done based on the mechanism of splicing from the extein. The intein and the first C-extein amino acid together act as a single turnover enzyme that splices the intein from the precursor protein. (Tori et al., 2010)  

To ease comparison between inteins, the residues in intein precursors have an existing numbering system: N terminus of the intein starts at 1 and continues sequentially to the C-terminus of the intein. All C-extein residues have a plus sign, starting with +1 from the N-terminus of the C-extein (Kazuo Tori and Francine B. Perler, 2011).  

All known inteins possess four conserved motifs, either called A, B, F, and G or N1, N3, C2, and C1 (Pietrokovski, S., 1994). Conserved residues can also be referred to using the block letter and the block position separated by a comma.  

The mech­an­isms of in­tein-me­di­ated pro­tein spli­cing 

Protein splicing is a rapid reaction; thus, the precursor protein is observed in native systems only rarely. Splicing rates or efficiency can alternate, depending on the exteins. The intein with the first C-extein residue together contain sufficient information for splicing in foreign proteins. (Perler, 2005) 

Standard protein splicing consists of 4 nucleophilic displacements by the 3 conserved splice junction residues. More detailed mechanisms of splicing are described further on under the three classes of inteins. The active intein sites are formed by folding the intein within the precursor, resulting in splice junctions and assisting internal intein residues being brought together (Perler, 2005).  

C-terminal cleavage and Asn cyclization are assisted by the intein penultimate His in Block G (Xu, M. Q. and Perler, 1996) by hydrogen bond to the Asn carbonyl oxygen, resulting in a more labile peptide (Duan et al., 1997).  

Block B’s Thr and His assist in acyl rearrangement at the N-terminal splice junction (Kawasaki et al., 1997) by hydrogen bonding to main chain atoms and holding the residue preceding the intein in a non-standard cis conformation or in a strained conformation (Poland et al., 2000). This indicates that any residue with capability to form similar hydrogen bonds can possibly substitute for these conserved residues.   

Class 1 in­tein

Most inteins fall into the first class of inteins and follow this standard protein splicing pathway of 4 nucleophilic displacement reactions. 

STEP 1:  The N-terminal splice junction is activated by a (S/O)-N acyl shift 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.  

STEP 2: Transesterification reaction: The upstream ester/thioester bond is attacked 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 formed branch is resolved by Cyclization of the conserved intein C-terminal Asn which forms a succinimide ring, resulting in cleavage of the C-terminal splice junction.  

STEP 4: A spontaneous (S/O)-N acyl rearrangement results in formation of a native peptide bond between the exteins.  

Additionally, a 5th step consists of the slow hydrolysis of succinimide, to form Asn or isoasparagine. (Beyer et al., 2019)  

The splicing mechanism of class 1 intein

The splicing mechanism of class 1 inteins

 

Class 2 in­tein

Class 2 inteins lack a N-terminal Ser, Thr or Cys, so they cannot perform the acyl shift that initiates the splicing reaction in the standard protein splicing pathway. Instead, Cys1 directly attacks an amide bond at the N-terminal splice junction to form the standard branched intermediate. Rest of the class 2 intein splicing reaction follows the same pathway class 1 inteins. (Kazuo Tori and Francine B. Perler, 2011)  

Known examples of Class 2 inteins are the Methanococcus jannaschii KlbA inteins, which have been identified with a N-terminal Ala1 instead of a Ser, Thr or Cys. (Saleh et al., 2011)  

STEP 1: Nucleophilic displacement: splicing is initiated by a direct attack on amide bond at the N-terminal splice junction by Cys1. Branched intermediate is formed.  

STEP 2: The formed branch is resolved by Cyclization of the conserved intein C-terminal Asn which forms a succinimide ring, resulting in cleavage of the C-terminal splice junction.  

STEP 3:  Spontaneous (S/O)-N acyl rearrangement results in formation of a native peptide bond between the exteins.  

 A few inteins have been identified with a C-terminal Gln (Q); 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. (Kazuo Tori and Francine B. Perler, 2011)​ 

The splicing mechanism by class 2 intein

The splicing mechanism of class 2 inteins

 

Class 3 in­tein

Similar to class 2 inteins, class 3 inteins lack a N-terminal Ser, Thr or Cys as well. Class 3 inteins differ from class 2 with their tendency to have a non-contiguous WCT motif consisting of Trp, Cys and Thr. Therefore class 3 inteins cannot initiate the splicing reaction by performing a acyl shift in the standard way. (Kazuo Tori and Francine B. Perler, 2011)  

Splicing mechanism of class 3 inteins includes 4 nucleophilic displacement reactions, and it was first defined using the Mycobacteriophage Bethlehem DnaB intein and the Deinococcus Radiodurans Snf2 intein. (Brace et al., 2010; Tori et al., 2010)  

STEP 1: Initiation of the splicing reaction: N-terminal splice junction peptide bond is targeted by a nucleophilic attack, the Cys from the WCT motif. The result is the formation of branched, thiol labile intermediate.  

STEP 2: The N-extein is transferred to the side chain of the +1 residue by a transesterification reaction. This results in the formation of the same branched intermediate as in class 1 intein reaction. After this step, the reaction follows the same pathway as the standard inteins (class 1).  

STEP 3: The formed branch is resolved by Cyclization of the conserved intein C-terminal Asn which forms a succinimide ring, resulting in cleavage of the C-terminal splice junction.  

STEP 4: A spontaneous (S/O)-N acyl rearrangement results in formation of a native peptide bond between the exteins. (Kazuo Tori and Francine B. Perler, 2011)  

The splicing mechanism of class 3 intein

The splicing mechanism of class 3 inteins