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Protein trans-splicing catalyzed by split inteins has been used for segmental isotopic labeling of proteins for alleviating the complexity of NMR signals. Whereas inteins spontaneously trigger protein splicing upon protein folding, inteins from extremely halophilic organisms require a high salinity condition to induce protein splicing. We designed and created a salt-inducible intein from the widely used DnaE intein from Nostoc punctiforme by introducing 29 mutations, which required a lower salt concentration than naturally occurring halo-obligate inteins. We determined the NMR solution structure of the engineered salt-inducible DnaE intein in 2 M NaCl, showing the essentially identical three-dimensional structure to the original one, albeit it unfolds without salts. The NMR structure of a halo-obligate intein under high salinity suggests that the stabilization of the active folded conformation is not a mere result of various intramolecular interactions but the subtle energy balance from the complex interactions, including the solvation energy, which involve waters, ions, co-solutes, and protein polypeptide chains., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans -peptidyl and trans -esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues.

Inteins catalyze self-excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the -1 and +2 positions, often strongly influence the protein-splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed "QuickDrop"-cassette mutagenesis for investigating the influences of 20 amino acid types at the -1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure-function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Protein trans-splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. We solved the 1.0 Å-resolution crystal structure of a fused variant from the naturally split gp41-1 intein, previously identified from environmental metagenomic sequence data. The structure of the 125-residue gp41-1 intein revealed a compact pseudo-C2-symmetry commonly found in the Hedgehog/Intein superfamily with extensive charge-charge interactions between the split N- and C-terminal intein fragments that are common among naturally occurring split inteins. We successfully created orthogonal split inteins by engineering a similar charge network into the same region of a cis-splicing intein. This strategy could be applicable for creating novel natural-like split inteins from other, more prevalent cis-splicing inteins. DATABASE: Structural data are available in the RCSB Protein Data Bank under the accession number 6QAZ., (© 2019 Federation of European Biochemical Societies.)

Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly conserved insertion site. A purifying selection mechanism directing the location of the HEN insertion site has not yet been identified. In this work, we solved the three-dimensional crystal structures of two inteins inserted in the cell division control protein 21 of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii. A comparison between the structures provides the structural basis for the thermo-stabilization mechanism of inteins that have lost the HEN domain during evolution. The presence of an entire extein domain in the intein structure from Pyrococcus horikoshii suggests the selection mechanism for the highly conserved HEN insertion point.

Inteins are mobile genetic elements that are spliced out of proteins after translation. Some inteins contain a homing endonuclease (HEN) responsible for their propagation. Hedgehog/INTein (HINT) domains catalyzing protein splicing and their nested HEN domains are thought to be functionally independent because of the existence of functional mini-inteins without HEN domains. Despite the lack of obvious mutualism between HEN and HINT domains, HEN domains are persistently found at one specific site in inteins, indicating their potential functional role in protein splicing. Here we report crystal structures of inactive and active mini-inteins derived from inteins residing in the transcription factor IIB of Methanococcus jannaschii and Methanocaldococcus vulcanius, revealing a novel modified HINT fold that might provide new insights into the mutualism between the HEN and HINT domains. We propose an evolutionary model of inteins and a functional role of HEN domains in inteins., (Copyright © 2017. Published by Elsevier Ltd.)

Intervening protein sequences (inteins) from extremely halophilic haloarchaea can be inactive under low salinity but could be activated by increasing the salt content to a specific concentration for each intein. The halo-obligatory inteins confer high solubility under both low and high salinity conditions. We showed the broad utility of salt-dependent protein splicing in cis and trans by demonstrating backbone cyclization, self-cleavage for purification, and scarless protein ligation for segmental isotopic labeling. Artificially split MCM2 intein derived from Halorhabdus utahensis remained highly soluble and was capable of protein trans-splicing with excellent ligation kinetics by reassembly under high salinity conditions. Importantly, the MCM2 intein has the active site residue of Ser at the +1 position, which remains in the ligated product, instead of Cys as found in many other efficient split inteins. Since Ser is more abundant than Cys in proteins, the novel split intein could widen the applications of segmental labeling in protein NMR spectroscopy and traceless protein ligation by exploiting a Ser residue in the native sequences as the +1 position of the MCM2 intein. The split halo-obligatory intein was successfully used to demonstrate the utility in NMR investigation of intact proteins by producing segmentally isotope-labeled intact TonB protein from Helicobacter pylori., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Protein splicing in trans by split inteins has increasingly become a powerful protein-engineering tool for protein ligation, both in vivo and in vitro. Over 100 naturally occurring and artificially engineered split inteins have been reported for protein ligation using protein trans-splicing. Here, we review the current status of the reported split inteins in order to delineate an empirical or rational strategy for constructing new split inteins suitable for various applications in biotechnology and chemical biology., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Protein splicing is an autocatalytic process involving self-excision of an internal protein domain, the intein, and concomitant ligation of the two flanking sequences, the exteins, with a peptide bond. Protein splicing can also take place in trans by naturally split inteins or artificially split inteins, ligating the exteins on two different polypeptide chains into one polypeptide chain. Protein trans-splicing could work in foreign contexts by replacing the native extein sequences with other protein sequences. Protein ligation using protein trans-splicing increasingly becomes a useful tool for biotechnological applications such as semi-synthesis of proteins, segmental isotopic labeling, and in vivo protein engineering. However, only a few split inteins have been successfully applied for protein ligation. Naturally split inteins have been widely used, but they are cross-reactive to each other, limiting their applications to multiple-fragment ligation. Based on the three-dimensional structures including two newly determined intein structures, we derived 21 new split inteins from four highly efficient cis-splicing inteins, in order to develop novel split inteins suitable for protein ligation. We systematically compared trans-splicing of 24 split inteins and tested the cross-activities among them to identify orthogonal split intein fragments that could be used in chemical biology and biotechnological applications.

Protein sequences are diversified on the DNA level by recombination and mutation and can be further increased on the RNA level by alternative RNA splicing, involving introns that have important roles in many biological processes. The protein version of introns (inteins), which catalyze protein splicing, were first reported in the 1990s. The biological roles of protein splicing still remain elusive because inteins neither provide any clear benefits nor have an essential role in their host organisms. We now report protein alternative splicing, in which new protein sequences can be produced by protein recombination by intermolecular domain swapping of inteins, as elucidated by NMR spectroscopy and crystal structures. We demonstrate that intein-mediated protein alternative splicing could be a new strategy to increase protein diversity (that is, functions) without any modification in genetic backgrounds. We also exploited it as a post-translational protein conformation-driven switch of protein functions (for example, as highly specific protein interference).

In protein splicing, an intervening protein sequence (intein) in the host protein excises itself out and ligates two split host protein sequences (exteins) to produce a mature host protein. Inteins require the involvement for the splicing of the first residue of the extein that follows the intein (which is Cys, Ser, or Thr). Other extein residues near the splicing junctions could modulate splicing efficiency even when they are not directly involved in catalysis. Mutual interdependence between this molecular parasite (intein) and its host protein (exteins) is not beneficial for intein spread but could be advantageous for intein survival during evolution. Elucidating extein-intein dependency has increasingly become important since inteins are recognized as useful biotechnological tools for protein ligation. We determined the structures of one of inteins with high splicing efficiency, the RadA intein from Pyrococcus horikoshii (PhoRadA). The solution NMR structure and the crystal structures elucidated the structural basis for its high efficiency and directed our efforts of engineering that led to rational design of a functional minimized RadA intein. The crystal structure of the minimized RadA intein also revealed the precise interactions between N-extein and the intein. We systematically analyzed the effects at the -1 position of N-extein and were able to significantly improve the splicing efficiency of a less robust splicing variant by eliminating the unfavorable extein-intein interactions observed in the structure. This work provides an example of how unveiling structure-function relationships of inteins offer a promising way of improving their properties as better tools for protein engineering., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Protein splicing catalyzed by inteins has enabled various biotechnological applications such as protein ligation. Successful applications of inteins are often limited by splicing efficiency. Here, we report the comparison of protein splicing between 20 different inteins from various organisms in identical contexts to identify robust inteins with foreign exteins. We found that RadA intein from Pyrococcus horikoshii and an engineered DnaB intein from Nostoc punctiforme demonstrated an equally efficient splicing activity to the previously reported highly efficient DnaE intein from Nostoc punctiforme. The newly identified inteins with efficient cis-splicing activity can be good starting points for the further development of new protein engineering tools., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

DnaE intein from Nostoc punctiforme (Npu) is one of naturally occurring split inteins, which has robust protein splicing activity. Highly efficient trans-splicing activity of NpuDnaE intein could widen various biotechnological applications. However, structural basis of the efficient protein splicing activity is poorly understood. As a first step toward better understanding of protein trans-splicing mechanism, we present the backbone and side-chain resonance assignments of a single chain variant NpuDnaE intein as determined by triple resonance experiments with [(13)C,(15)N]-labeled protein.

Naturally split DnaE intein from Nostoc punctiforme (Npu) has robust protein trans-splicing activity and high tolerance of sequence variations at the splicing junctions. We determined the solution structure of a single chain variant of NpuDnaE intein by NMR spectroscopy. Based on the NMR structure and the backbone dynamics of the single chain NpuDnaE intein, we designed a functional split variant of the NpuDnaE intein having a short C-terminal half (C-intein) composed of six residues. In vivo and in vitro protein ligation of model proteins by the newly designed split intein were demonstrated.

Background: Protein trans-splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein trans-splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized., Principal Findings: We report the identification of new functional split sites in DnaE inteins from Synechocystis sp. PCC6803 and from Nostoc punctiforme. One of the newly engineered split intein bearing C-terminal 15 residues showed more robust protein trans-splicing activity than naturally occurring split DnaE inteins in a foreign context. During the course of our experiments, we found that protein ligation by protein trans-splicing depended not only on the splicing junction sequences, but also on the foreign extein sequences. Furthermore, we could classify the protein trans-splicing reactions in foreign contexts with a simple kinetic model into three groups according to their kinetic parameters in the presence of various reducing agents., Conclusion: The shorter C-intein of the newly engineered split intein could be a useful tool for biotechnological applications including protein modification, incorporation of chemical probes, and segmental isotopic labelling. Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein trans-splicing, which could significantly enhance the applications of protein ligation by protein trans-splicing.

Vint proteins have been identified in unicellular metazoans as a novel hedgehog‐related gene family, merging the von Willebrand factor type A domain and the Hedgehog/INTein (HINT) domains. We present the first three‐dimensional structure of the Vint domain from Tetrahymena thermophila corresponding to the auto‐processing domain of hedgehog proteins, shedding light on the unique features, including an adduct recognition region (ARR). Our results suggest a potential binding between the ARR and sulfated glycosaminoglycans like heparin sulfate. Moreover, we uncover a possible regulatory role of the ARR in the auto‐processing by Vint domains, expanding our understanding of the HINT domain evolution and their use in biotechnological applications. Vint domains might have played a crucial role in the transition from unicellular to multicellular organisms. [ABSTRACT FROM AUTHOR]

Protein splicing in trans by split inteins has increasingly become a powerful protein-engineering tool for protein ligation, both in vivo and in vitro. Over 100 naturally occurring and artificially engineered split inteins have been reported for protein ligation using protein trans-splicing. Here, we review the current status of the reported split inteins in order to delineate an empirical or rational strategy for constructing new split inteins suitable for various applications in biotechnology and chemical biology. [ABSTRACT FROM PUBLISHER]

Protein splicing in trans by split inteins has become a useful tool for protein engineering in vivo and in vitro. Inteins require Cys, Ser or Thr at the first residue of the C-terminal flanking sequence because a thiol or hydroxyl group in the side chains is a nucleophile indispensable for the trans-esterification step during protein splicing. Newly-identified distinct sequences with homology to the hedgehog/intein superfamily, called bacterial intein-like ( BIL) domains, often do not have Cys, Ser, or Thr as the obligatory nucleophilic residue found in inteins. We demonstrated that BIL domains from Clostridium thermocellum ( Cth) are proficient at protein splicing without any sequence changes. We determined the first solution NMR structure of a BIL domain, Cth BIL4, to guide engineering of split BIL domains for protein ligation. The newly-engineered split BIL domain could catalyze protein ligation by trans-splicing. Protein ligation without any nucleophilic residues of Cys, Ser and Thr could alleviate junction sequence requirements for protein trans-splicing imposed by split inteins and could broaden applications of protein ligation by protein trans-splicing. Database The resonance assignments and structure coordinates have been deposited in BMRB (18653) and RCSB (2LWY) [ABSTRACT FROM AUTHOR]

The RadA intein from the hyperthermophilic archaebacterium Pyrococcus horikoshii was cloned, expressed and purified for subsequent structure determination. The protein crystallized rapidly in several conditions. The best crystals, which diffracted to 1.75 Å resolution, were harvested from drops consisting of 0.1 M HEPES pH 7.5, 3.0 M NaCl and were cryoprotected with Paratone-N before flash-cooling. The collected data were processed in the orthorhombic space group P212121, with unit-cell parameters a = 58.1, b = 67.4, c = 82.9 Å. Molecular replacement with Rosetta using energy- and density-guided structure optimization provided the initial solution, which is currently under refinement. [ABSTRACT FROM AUTHOR]