Intermolecular domain swapping induces intein-mediated protein alternative splicing

Aranko, A. Sesilja; Oeemig, Jesper S.; Kajander, Tommi; Iwaï, Hideo

doi:10.1038/nchembio.1320

Cited by 50 publications

(53 citation statements)

References 51 publications

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“…Splicing reaction mixtures were incubated for 10 min at 63°C, separated by SDS-PAGE (8%-16%), and stained with Coomassie, and the levels of precursor (P), ligated exteins (LEs), and intein (I) were measured by densitometry. The intein appears as a dimer (×2), consistent with previous observations (Oeemig et al 2012;Aranko et al 2013). (TE) Tris-EDTA, the storage buffer for ssDNA; (ssDNA) M13mp18 ssDNA; (dNTPs) deoxynucleotide triphosphates; (dsDNA) double-stranded plasmid DNA; (RNA) total cellular RNA.…”

Section: Resultssupporting

confidence: 71%

Protein splicing of a recombinase intein induced by ssDNA and DNA damage

2016

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Inteins (or protein introns) autocatalytically excise themselves through protein splicing. We challenge the longconsidered notion that inteins are merely molecular parasites and posit that some inteins evolved to regulate host protein function. Here we show substrate-induced and DNA damage-induced splicing, in which an archaeal recombinase RadA intein splices dramatically faster and more accurately when provided with ssDNA. This unprecedented example of intein splicing stimulation by the substrate of the invaded host protein provides compelling support in favor of inteins acting as pause buttons to arrest protein function until needed; then, an immediate activity switch is triggered, representing a new form of post-translational control.

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Section: Resultssupporting

confidence: 71%

Protein splicing of a recombinase intein induced by ssDNA and DNA damage

2016

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“…3 A and B and SI Appendix, Figs. S5 and S6) (17). This shows that the greatest values of Δδ are present in residues structurally proximal to the loop, with modest perturbations elsewhere.…”

Section: Resultsmentioning

confidence: 87%

“…NMR spectroscopy was performed at 37°C in field strengths of 600 or 900 MHz on the uniformly 13 (36). The chemical shift perturbation values were then calculated and represented as a heat map on the crystal structure of Npu DnaE (PDB ID: 4kl5) (17).…”

Section: Methodsmentioning

confidence: 99%

A promiscuous split intein with expanded protein engineering applications

Stevens

Sekar

Shah

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

117

119

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The protein trans-splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and biotechnology. However, currently used naturally split inteins suffer from an "extein dependence," whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods. To address this, we describe a mechanism-guided protein engineering approach that imbues ultrafast DnaE split inteins with minimal extein dependence. The resulting "promiscuous" inteins are shown to be superior reagents for protein cyclization and protein semisynthesis, with the latter illustrated through the modification of native cellular chromatin. The promiscuous inteins reported here thus improve the applicability of existing PTS methods and should enable future efforts to engineer promiscuity into other naturally split inteins. A n intein is an intervening protein domain that undergoes a unique posttranslational autoprocessing event, termed protein splicing. In this spontaneous process, the intein excises itself from the host protein and, in the process, ligates together the flanking N-and C-terminal residues (exteins) to form a native peptide bond (SI Appendix, Fig. S1A) (1, 2). Although inteins are most frequently found as a contiguous domain, some exist in a naturally split form. In this case, the two fragments are expressed as separate polypeptides and must associate before splicing takes place, so-called protein trans-splicing (SI Appendix, Fig. S1B). Unlike many well-characterized contiguous inteins that splice slowly, several naturally split inteins demonstrate rapid splicing kinetics (3-6). Indeed, the discovery of these ultrafast split inteins has enabled the development of numerous tools for both synthetic and biological applications (1).A major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site. In addition to a mandatory catalytic Cys, Ser, or Thr residue at position +1 (i.e., the first residue within the C-extein), there is a bias for residues resembling the proximal N-and C-extein sequence found in the native insertion site. Deviation from this preferred sequence context leads to a marked reduction in splicing activity, limiting the applicability of protein trans-splicing (PTS)-based methods (3, 7-11). Accordingly, there is a need for split inteins whose activities are minimally affected by local sequence environment. Although efforts have previously been made to engineer promiscuous inteins (12, 13), these have not focused on naturally split inteins, which have superior fragment association and splicing kinetics (4-6).Here, we report engineered versions of naturally split inteins that possess greatly improved extein tolerance. Guided by our understanding of active site interactions critical for efficient protein splicing, we carried out targeted saturation mutagenesis of an ultrafast split intein and then used a cell-based selection system to...

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“…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.…”

Section: The Intein Foldmentioning

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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Intermolecular domain swapping induces intein-mediated protein alternative splicing

Cited by 50 publications

References 51 publications

Protein splicing of a recombinase intein induced by ssDNA and DNA damage

Protein splicing of a recombinase intein induced by ssDNA and DNA damage

A promiscuous split intein with expanded protein engineering applications

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

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