Highly Efficient and More General cis- and trans-Splicing Inteins through Sequential Directed Evolution

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...

Section: Distal Mutations Affect Protein Splicingmentioning

confidence: 90%

Section: Distal Mutations Affect Protein Splicingmentioning

confidence: 99%

Section: Distal Mutations Affect Protein Splicingmentioning

confidence: 99%

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Structural and Dynamical Features of Inteins and Implications on Protein Splicing

Eryilmaz

Shah

Muir

et al. 2014

“…Split inteins hold a tremendous potential for a wide range of protein engineering approaches (17,19,30). Recently, novel biotechnological applications for split inteins were described.…”

Section: Discussionmentioning

confidence: 99%

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

Carvajal-Vallejos

Pallisse

Mootz

et al. 2012

Self Cite

142

186

Background: Hypothetical natural split inteins from environmental metagenomic sources were evaluated experimentally for the first time. Results: Four inteins showed the highest known reaction rates and efficiencies for protein trans-splicing and could be used efficiently for C-cleavage. Conclusion: These inteins pushed the catalytic limit of protein trans-splicing, although this was unpredictable from their primary sequence. Significance: These inteins hold great potential as versatile protein engineering tools.

“…The small size of this fragment of 37 or 28 aa also calls for applications using semisynthetic protein trans-splicing (23,25,51,52).…”

Section: Discussionmentioning

confidence: 99%

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

Bachmann

Mootz

2015

Self Cite

Background: Intein-mediated protein splicing involves rearrangement of the polypeptide backbone through ester intermediates and excises the intein from the precursor. Results: An unusual split intein was identified that converts an oxoester into a thioester. Conclusion: Inteins can catalyze two consecutive formally uphill reactions. Significance: The first example of this special case in protein splicing underlines the mechanistic diversity of inteins.