Kinetic Analysis of the Individual Steps of Protein Splicing for the Pyrococcus abyssi PolII Intein

Abstract

Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed and purified as an unspliced precursor, which allows for a detailed in vitro kinetic analysis of the individual steps of protein splicing. The first order rate constant for splicing of this intein, which has a noncanonical Gln at its C terminus, is 9.3 ؋ 10 ؊6 s ؊1 at 60°C. The rate constant for splicing increases 3-fold with substitution of Asn for the C-terminal Gln. The pseudo first order rate constant of dithiothreitol-dependent N-terminal cleavage is 1 ؋ 10 ؊4 s ؊1 . The first order rate constant of C-terminal cleavage is 1.2 ؋ 10 ؊5 s ؊1 with Gln at the C-terminal position, 2.8 ؋ 10 ؊4 s ؊1 with Asn, and decreases significantly with mutation of the penultimate His of the intein to Ala. N-terminal cleavage is most efficient between pH 7 and 7.5 and decreases at both more acidic and alkaline pH values, whereas C-terminal cleavage and splicing are both efficient over a broader range of pH values.Protein splicing involves the post-translational, self-directed excision of an intervening polypeptide, or intein, from flanking polypeptides, or exteins. The splicing process also involves the specific ligation of the extein segments by an amide bond, concomitant with the excision of the intein (1).The canonical mechanism of protein splicing consists of four separate but coordinated chemical steps each directed by the intein without the assistance of other proteins or cofactors (1). The first step of splicing is an N-S or N-O acyl rearrangement of the amide bond linking the N-extein and intein (see Fig. 1, step 1). This is followed by a transesterification that transfers the N-extein to the side chain of the N-terminal Cys, Ser, or Thr of the C-extein (see Fig. 1, step 2). The third step of splicing couples the cyclization of the C-terminal Asn of the intein to peptide bond cleavage, and excises the intein from the newly ligated exteins (see Fig. 1, step 3). Finally, the ester bond linking the exteins is rearranged to an amide (see Fig. 1, step 4), and the C-terminal aminosuccinimide of the intein is presumably hydrolyzed to either Asn or iso-Asn (see Fig. 1, step 5).Inteins that splice via variations of the canonical mechanism have previously been described. For instance, our group recently described the protein splicing of the intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi (Pab), 1 which is able to facilitate protein splicing with a Cterminal Gln residue (2). These results are complementary to those of Pietrokovski and coworkers (3), who demonstrated that the intein that interrupts the ribonucleotide reductase (RNR) of the Chilo iridescent virus can facilitate protein splicing with a C-terminal Gln and that the intein interrupting the RNR of Carboxydothermus hydrogenoformans can facilitate protein splicing w...

Recent progress in intein research: from mechanism to directed evolution and applications

“…Other major challenges for a better molecular characterization of inteins include the understanding of the extein sequence dependence, not only on the level of the primary sequence flanking the intein but also in the context of the protein 3-D structure and folding pathway. An intriguing open question is also how conformational changes are brought about within the intein structure during splicing, i.e., to facilitate the trans-esterification step, as has been proposed numerous times in the literature [23,35,36,38,40,55,56,58,[135][136][137]. In general, we believe that a deeper understanding of the different protein splicing mechanisms, as well as new means to control or circumvent the contextdependency of inteins, for example with the use of evolved super-mutants, will greatly strengthen the applicability of the many intein-related technologies to challenges in protein engineering and protein biotechnology.…”

Protein Splicing: How Inteins Escape from Precursor Proteins

“…Alternatively, given that C-terminal cleavage is favored at low pH (65, 70, 71), protonation of the backbone amide nitrogen of the scissile peptide bond may have precedence over deprotonation of the Asn side-chain amide (72). Separate studies suggest two other modes of catalysis: change in the local environment near the scissile bond that depends on branched ester formation (69) and destabilization of the scissile bond by a polarizable adjacent C-extein residue (73).…”

“…Some inteins lacking Asn G:7 have similar residues (Asp and Gln) that can undergo cyclization to cleave the C-terminal splice site (20, 71, 78–80). For both the Pyrococcus abyssi and the Methanoculleus marisnigri Pol II inteins, splicing with a C-terminal Gln is slow, but is improved with substitution to Asn (71, 78, 79).…”

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

“…Asp and Gln can undergo similar cyclization reactions. 6,7,[9][10][11] The intein penultimate His assists Asn cyclization. 6,7 A native peptide bond is formed between the exteins (VI) after a spontaneous [S/O]-N acyl shift.…”