An alternative protein splicing mechanism for inteins lacking an N-terminal nucleophile

Abstract

Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identi®ed. The conserved Ser or Cys at the intein N-terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since 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. Despite the presence of an N-terminal Ala and a penultimate Ser, the KlbA inteins splice ef®ciently using an alternative protein splicing mechanism. In this non-canonical pathway, the C-extein nucleophile 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 the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins.

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

“…These results were similar to those observed after mutation of the Mja KlbA (Ala1) and MP-Be DnaB (Pro1) intein Ntermini in that some mutations reduced the reactivity of the N-terminal splice junction, while others did not. 5,12,13 However, the relative amounts of products observed with each amino acid were usually specific to the intein, which is also true for all positions tested in this study.…”

“…[1][2][3][4][5] Two variations in the intein-mediated protein splicing mechanism were previously described, which involve direct attack on an amide bond at the N-terminal splice junction to generate a BI. [5][6][7]12,13 In Class 2 inteins, exemplified by the Methanococcus jannaschii (Mja) KlbA intein, 12,13 the Block G BI (III) is formed by nucleophilic attack of Cysþ1 on the peptide bond at the N-terminal splice junction (Fig. 2).…”

“…The B:10 His is essential for splicing and N-terminal cleavage, while mutation of the more variable B:7 residue in Class 1 and 2 inteins usually reduces splicing and N-terminal cleavage, but does not block it. [5][6][7][12][13][14] The essential characteristics of Class 3 inteins are the mechanistic properties specific to this subfamily of inteins as determined in the prototypic MP-Be DnaB intein: (a) cleavage at the N-terminal splice junction in the absence of all standard Class 1 and Class 2 N-and C-terminal splice junction nucleophiles at A:1, G:7, and G:8, (b) activation of the Nterminal splice junction by a Block B motif that includes the conserved His (B:10) and the WCT triplet Trp (B:12), (c) breakdown of native but not denatured Block G BI (III) in response to thiols or Cys despite an ester linkage at the Block G branch point, (d) an absolute requirement of the WCT triplet Cys at F:4 for splicing, (e) both a Block F BI (VIII) and a Block G BI (III), and (f) nonconservative mutation of the F:4 Cys prevents formation of both the Block F BI and the Block G BI, as well as off-pathway N-terminal cleavage.…”

Inteins in Microbial Genomes: Distribution, Mechanism, and Function

“…Polymorphisms in nucleophiles and conserved residues have become more evident as more inteins have been sequenced. A second protein splicing mechanism has been determined for inteins lacking an N-terminal nucleophile [5].…”

Protein Splicing Mechanisms and Applications

“…Analysis of the Methanococcus jannaschii (Mja) KlbA intein showed that (a) it spliced efficiently in Escherichia coli, (b) included the standard branched intermediate, (c) Cys + 1 was required for splicing and thiol-induced N-terminal cleavage of the branched intermediate, (d) the intein C-terminal Asn was required for C-terminal cleavage, (e) the Block B Thr and His activated the N-terminal splice junction, and (f) the standard linear thioester was observed after 'reversion' of Ala1 to Cys (15). These data support a 3-step mechanism in which Cys + 1 directly attacks an amide bond at the N-terminal splice junction resulting in formation of the normal branched intermediate.…”