Chemical Bypass of Intein‐Catalyzed N–S Acyl Shift in Protein Splicing

Abstract

Inteins are self-processing protein domains that excise themselves out of a precursor polypeptide chain in a multistep pathway termed protein splicing. In the course of this reaction, the sequences flanking the intein, termed N-and Cterminal exteins, are linked with a peptide bond (Scheme 1 A). Inteins perform only a single turn-over, however, they employ catalytic strategies similar to those of enzymes. While inteins have found widespread use in many applications in biotechnology and protein chemistry, important aspects of the mechanism of protein splicing are still not understood. [1] In particular, the N-S (or N-O) acyl shift of the upstream scissile peptide bond into a thioester (or oxoester) remains intriguing (Scheme 1 B). This rearrangement represents the first step of protein splicing in standard inteins [2] and is widely exploited for the generation of protein thioesters. [3] Although thermodynamically unfavored, it does not require any cofactors or energy sources. Several catalytic mechanisms have been proposed for this reaction, including destabilization of the ground state of the scissile peptide bond, general acid-base catalysis to increase the nucleophilicity of the cysteine (or serine) side chain at position 1 of the intein, and stabilization of the tetrahedral intermediate by means of an oxyanion hole (Scheme 1 B). Some of these mechanisms, if not all, can likely be used in combination and may contribute to catalysis to different degrees in different inteins. Here, we pursued a novel chemical approach to directly probe the importance of ground-state destabilization by introducing an alkyl substituent at the amide nitrogen of the scissile peptide bond (Scheme 1 B); a strategy inspired by the recent development of N-S switch devices for the chemical synthesis of peptide thioesters. [4] This chemical manipulation indeed supported the N-S acyl shift, even to the extent that it could complement an otherwise essential part of the catalytic framework of the intein, the highly conserved block B histidine. Together, our findings reveal the role of the histidine in a ground-state destabilization mechanism and rule out other roles previously proposed.The ground-state destabilization of the upstream scissile peptide bond has been proposed on the basis of structural analyses, but its direct investigation remains challenging. In two intein structures, deviations of this peptide bond from the common trans conformation to the energetically less favored cis conformation [5] or a distorted conformation [6] were observed (Scheme 1 B). A solution NMR study by Muir and co-workers on an Mxe GyrA intein precursor that is active in the N-S acyl shift suggested a significant distortion of the peptide bond. [7] Importantly, the highly conserved histidine residue in the block B signature motif (His 75 im Mxe GyrA Intein) was essential for this effect, as a His75Ala mutant was inactive in thioester formation and did not induce any measurable conformational strain on the peptide bond. [7] Block B is one of the conserved seq...

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

“…We also prepared the construct Int C (H90A,N152A,Cϩ1A)-Trx-His 6 , in which the highly conserved histidine of block B was mutated. This histidine was shown in other inteins to be important or essential for (thio)ester formation at the N-terminal scissile bond (30,(37)(38)(39). No cleavage could be observed in combination with the wild-type Int N construct (data not shown).…”

“…Peptide Synthesis and Purification-Solid phase peptide synthesis and purification was performed as reported previously (29,30) …”

From protein total synthesis to peptide transamidation and metathesis: playing with the reversibility of N,S-acyl or N,Se-acyl migration reactions

“…Inteins remove themselves from a precursor protein and concomitantly form a peptide bond between the flanking protein sequences, termed as N-terminal and C-terminal exteins. The mechanism of protein splicing is still in debate but conformational strain of the scissile peptide bond to cysteine induced by the intein protein is thought to play an important role [33,34 ].…”

Reversible Native Chemical Ligation: A Facile Access to Dynamic Covalent Peptides

“…36 More recently, N-(methyl)-cysteine has also been shown to promote the N → S acyl shift in the mechanistic studies of model inteins. 22 Interestingly, N-(methyl)-cysteine is a nonribosomal amino acid that can be found in natural products. 37 N-Methylation is also a common post-translational modification taking place in many microbial peptides such as cyclosporins.…”

“…Interestingly, and although NCL is not a chemical reaction involved in any biochemical process, a mechanism similar to a reverse NCL reaction (retro-NCL) is found in the self-splicing of inteins. 21−23 It is proposed that, in the folded intein, the scissile bond at the cysteine residue is preferentially in a cis or distorted conformation favoring the intramolecular nucleophilic attack of the cysteine thiol 21,22 (N → S acyl shift, Figure 1a) with an equilibrium constant 4000 times larger than that of the corresponding trans conformation. 23 In synthetic chemistry, peptide thioesters have been prepared by fragmentation of Cterminated cysteine peptides using the retro-NCL, but under harsh acidic conditions and in the presence of a large excess of an aliphatic thiol.…”