A conserved threonine spring‐loads precursor for intein splicing

Abstract

Protein splicing is an autocatalytic process where an ''intein'' self-cleaves from a precursor and ligates the flanking N-and C-"extein'' polypeptides. Inteins occur in all domains of life and have myriad uses in biotechnology. Although the reaction steps of protein splicing are known, mechanistic details remain incomplete, particularly the initial peptide rearrangement at the N-terminal extein/intein junction. Recently, we proposed that this transformation, an N-S acyl shift, is accelerated by a localized conformational strain, between the intein's catalytic cysteine (Cys1) and the neighboring glycine (Gly-1) in the N-extein. That proposal was based on the crystal structure of a catalytically competent trapped precursor. Here, we define the structural origins and mechanistic relevance of the conformational strain using a combination of quantum mechanical simulations, mutational analysis, and X-ray crystallography. Our results implicate a conserved, but largely unstudied, threonine residue of the Ssp DnaE intein (Thr69) as the mediator of conformational strain through hydrogen bonding. Further, the strain imposed by this residue is shown to position the splice junction in a manner that enhances the rate of the N-S acyl shift substantially. Taken together, our results not only provide fundamental understanding of the control of the first step of protein splicing but also have important implications in various biotechnological applications that require precursor manipulation.

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

“…1A) has been seen in all intein structures solved by NMR and x-ray crystallography (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). The fold comprises primarily ␤-sheets, loops, and two short helices and has pseudo two-fold symmetry (Fig.…”

“…Additionally, some inteins lack a Block A nucleophile; instead they have an alanine or a proline. Several studies have shown a twisted or destabilized N-terminal scissile peptide bond in the precursor protein (5,23). These inteins directly form a typical branched intermediate upon N-terminal activation (24), or in some cases, they use a Block F cysteine to form a different branched (thio-)ester intermediate before the canonical branched intermediate (25).…”

An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site

“…Therefore, formation or stabilization of the Ser1-oxoester should require more catalytic assistance from the intein. The Thr or Ser at the conserved position of the Ser-87 in the GOS-TerL intein have previously been suggested to help in distorting the scissile peptide bond at the upstream splice junction to facilitate the N-S acyl shift (41). A similar role in the N-O shift as well as a role in preparing the oxoester for the transesterification reaction is conceivable for the GOS-TerL intein.…”

Backbone assignments of mini-RecA intein with short native exteins and an active N-terminal catalytic cysteine

“…It was found that the amine was highly polarized and that the peptide bond was scissile and interactions with the B-block histidine were implicated as the cause of the effect. Later work showed a similarly strained bond in this location (Callahan et al 2011), but implicated the nearby B-block threonine (Dearden et al 2013). …”

“…We therefore created a novel construct based on the minimized RecA intein, whose backbone assignments were previously reported (Du et al 2008), in which the B-block threonine is mutated to alanine. This mutation was shown to reduce strain between the first cysteine of the intein and the preceding N-extein residue with the effect of eliminating splicing and N-terminal cleavage (Dearden et al 2013). Additionally, the terminal asparagine is mutated to alanine to eliminate C-terminal cleavage.…”