Probing Intein-Catalyzed Thioester Formation by Unnatural Amino Acid Substitutions in the Active Site

Abstract

Inteins are single-turnover catalysts that splice themselves out of a precursor polypeptide chain. For most inteins, the first step of protein splicing is the formation of a thioester through an N-S acyl shift at the upstream splice junction. However, the mechanism by which this reaction is achieved and the impact of mutations in and close to the active site remain unclear on the atomic level. To investigate these questions, we have further explored a split variant of the Ssp DnaB intein by introducing substitutions with unnatural amino acids within the short synthetic N-terminal fragment. A previously reported collapse of the oxythiazolidine anion intermediate into a thiazoline ring was found to be specificially dependent on the methyl side chain of the flanking Ala(-1). The stereoisomer d-Ala and the constitutional isomers β-Ala and sarcosine did not lead to this side reaction but rather supported splicing. Substitution of the catalytic Cys1 with homocysteine strongly inhibited protein splicing; however, thioester formation was not impaired. These results argue against the requirement of a base to deprotonate the catalytic thiol group prior to the N-S acyl shift, because it should be misaligned for optimal proton abstraction. A previously described mutant intein evolved for more general splicing in different sequence contexts could even rather efficiently splice with this homocysteine. Our findings show the large impact of some subtle structural changes on the protein splicing pathway, but also the remarkable tolerance toward other changes. Such insights will also be important for the biotechnological exploitation of inteins.

Protein Splicing: How Inteins Escape from Precursor Proteins

“…However, studies of the Synechocystis sp. PCC6803 Ssp DnaB intein with an unnatural N-terminal residue indicate that activation of this nucleophile is not essential for linear thioester formation (62), emphasizing the unique active sites and catalytic strategies utilized by individual inteins.…”

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

“…In the Ssp DnaB mini-intein, analysis of the crystal structure explains why the Ϫ1 position can only be occupied by a Gly. Modeling suggests that any bigger residue would abrogate splicing by causing steric clashes (42) (Fig. 3B).…”

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

“…The idea that a base is required for deprotonation of the N-nucleophile in class 1 inteins, however, is challenged by a recent study that used unnatural amino acid substitutions to probe the formation of thioester bonds at the N-terminal splice junction of a semi-synthetic Ssp DnaB split intein [47]. Specifically, the introduction of homocysteine (Hcy) in place of the natural cysteine residue at position A:1 did not significantly decrease the rate of thioester hydrolysis, indicating that the N-S acyl shift was largely unaffected.…”

“…A so-far-underestimated facet of the N-X acyl shift may be the documented prevalence of aminoacyl-cysteinyl peptide bonds to spontaneously rearrange into the thioester [47], in particular under acidic conditions, when protonation of the a-amino group favors the equilibrium to the thioester. Efforts in the field of synthetic peptide chemistry to achieve new synthetic routes to peptide thioesters have exploited this rearrangement under acidic conditions and even at neutral pH.…”