Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Romanelli, Alessandra; Shekhtman, Alexander; Cowburn, David; Muir, Tom W.

doi:10.1073/pnas.0306616101

Cited by 158 publications

(141 citation statements)

References 40 publications

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“…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).…”

Section: N-to-o/s Acyl Shiftmentioning

confidence: 99%

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

Eryilmaz

Shah

Muir

et al. 2014

Journal of Biological Chemistry

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Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoelike pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of inteinbased technologies.Protein splicing is a posttranslational modification, a multistep autoprocessing event, where intervening proteins (inteins) self-excise themselves from the precursors followed by the ligation of external proteins (exteins) (1). Once thought anomalies, inteins have since been found in all domains of life. Beyond their biological context, inteins are particularly intriguing as their capacity to process the polypeptide backbone makes them useful tools for protein engineering. Despite the fact that all inteins share the same fold and have highly conserved sequence motifs in their active sites, inteins have surprisingly different splicing efficiencies, and their extein sequence preferences differ dramatically. Here, we review studies on intein structure and dynamics that shed light on their complex conserved fold and their divergent efficiency and substrate specificity. The Intein FoldThe conserved horseshoe-like fold of inteins (Fig. 1A) has been seen in all intein structures solved by NMR and x-ray crystallography (2-11). The fold comprises primarily ␤-sheets, loops, and two short helices and has pseudo two-fold symmetry (Fig. 1B). Given this symmetry, it has been proposed that this fold arose due to a gene duplication event of some parent protein (6). The intein fold has three remarkable features: 1) the topology is complex, involving multiple passes of the polypeptide chain back and forth between the symmetry-related halves (Fig. 1B); 2) the extein-bearing termini of the intein are brought in close proximity (Ͻ10 Å) for splicing; and 3) multiple protease-like active sites composed of conserved sequence motifs are built around these termini to carry out each of the chemical steps involved in protein splicing. Folding of an intein is coupled to the reaction; the initial structure facilitates the first step of splicing, and thereafter each splicing step causes local conformational changes, affecting the fold of the catalytic apparatus and hence affecting the reaction coordinate and shifting the equilibrium positi...

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Section: N-to-o/s Acyl Shiftmentioning

confidence: 99%

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

Eryilmaz

Shah

Muir

et al. 2014

Journal of Biological Chemistry

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“…These residues include the conserved His B:10 residue in all types of inteins (9,17) and the WCT motif Trp B:12 residue in class 3 inteins (2,20). The B:7 residue is not highly conserved but assists in N-terminal reactions, to various extents, in all classes of inteins (2,14,15,20).…”

Section: Splicing Of Wild-type Inteinsmentioning

confidence: 99%

“…Mutagenesis of conserved residues in block B. Residues in intein block B (Table 1) activate the N-terminal splice junction for both on-and off-pathway cleavage (2,9,10,12,17,19,20). These residues include the conserved His B:10 residue in all types of inteins (9,17) and the WCT motif Trp B:12 residue in class 3 inteins (2,20).…”

Section: Splicing Of Wild-type Inteinsmentioning

confidence: 99%

Expanding the Definition of Class 3 Inteins and Their Proposed Phage Origin

Tori

Perler

2011

J Bacteriol

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This study determined which class the mycobacteriophage Catera Gp206 and Nocardioides sp. JS614 TOPRIM inteins belong to based on catalytic mechanism. The mycobacteriophage Catera Gp206 intein (starting with Ser 1 ) is a class 3 intein, and its Ser 1 residue is not required for splicing. Based on phylogenetic analysis, we propose that class 3 inteins arose from a single mutated intein that was spread by phage into predominantly helicase genes in various phages and their hosts.

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“…6 We noted that Gly-1, which corresponds to the N-extein junction residue in the precursor, adopts an unusual geometry, which is consistent with similar studies involving splicing reactions in which distortions are present. [7][8][9][10][11] The (u,W) angles of Gly-1 fall outside the typical range for this residue. We speculated that the structural distortion at Gly-1 arises from hydrogen bonding with a conserved threonine, Thr69 [ Fig.…”

Section: Introductionmentioning

confidence: 99%

A conserved threonine spring‐loads precursor for intein splicing

et al. 2013

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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.

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Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Cited by 158 publications

References 40 publications

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

Expanding the Definition of Class 3 Inteins and Their Proposed Phage Origin

A conserved threonine spring‐loads precursor for intein splicing

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