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Hideo Iwai - One of the best experts on this subject based on the ideXlab platform.
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mini Intein structures from extremophiles suggest a strategy for finding novel robust Inteins
2021Co-Authors: Mimmu Karoliina Hiltunen, Hannes M. Beyer, Hideo IwaiAbstract:Inteins are prevalent among extremophiles. Mini-Inteins with robust splicing properties are of particular interest for biotechnological applications due to their small size. However, biochemical and structural characterization has still been limited to a small number of Inteins, and only a few serve as widely used tools in protein engineering. We determined the crystal structure of a naturally occurring Pol-II mini-Intein from Pyrococcus horikoshii and compared all three mini-Inteins found in the genome of P. horikoshii. Despite their similar sizes, the comparison revealed distinct differences in the insertions and deletions, implying specific evolutionary pathways from distinct ancestral origins. Our studies suggest that sporadically distributed mini-Inteins might be more promising for further protein engineering applications than highly conserved mini-Inteins. Structural investigations of additional Inteins could guide the shortest path to finding novel robust mini-Inteins suitable for various protein engineering purposes.
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the convergence of the hedgehog Intein fold in different protein splicing mechanisms
2020Co-Authors: Hannes M. Beyer, Alexander Wlodawer, Kornelia M. Mikula, Salla I Virtanen, Sesilja A Aranko, George T Lountos, O Samuli H Ollila, Hideo IwaiAbstract: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.
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the crystal structure of the naturally split gp41 1 Intein guides the engineering of orthogonal split Inteins from cis splicing Inteins
2020Co-Authors: Hannes M. Beyer, Alexander Wlodawer, Kornelia M. Mikula, Hideo IwaiAbstract:Protein trans-splicing catalyzed by split Inteins has increasingly become useful as a protein engineering tool. We solved the 1.0 A-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.
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Crystal structures of CDC21-1 Inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site
2019Co-Authors: Hannes M. Beyer, Kornelia M. Mikula, Tatiana V. Kudling, Hideo IwaiAbstract: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.
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the crystal structure of the naturally split gp41 1 Intein guides the engineering of orthogonal split Inteins from a cis splicing Intein
2019Co-Authors: Hannes M. Beyer, Alexander Wlodawer, Kornelia M. Mikula, Hideo IwaiAbstract:Protein trans-splicing catalyzed by split Inteins has increasingly become useful as a protein engineering tool. The 1.0 A-resolution crystal structure of a variant from naturally split gp41-1 Intein, identified from the environmental metagenomic sequence data, revealed an improved pseudo-C2-symmetry commonly found in the Hedgehog/Intein (HINT) superfamily with extensive charge-charge interactions between the split N-and C-terminal Intein fragments. We successfully created orthogonal split Inteins by engineering a similar charge network in the same region of a cis-splicing Intein. The same strategy could be applicable for creating novel natural-like split Inteins from other, more prevalent cis-splicing Inteins.
Tom W Muir - One of the best experts on this subject based on the ideXlab platform.
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an atypical mechanism of split Intein molecular recognition and folding
2018Co-Authors: Adam J. Stevens, Josef A. Gramespacher, David Cowburn, Giridhar Sekar, Tom W MuirAbstract:Split Inteins associate to trigger protein splicing in trans, a post-translational modification in which protein sequences fused to the Intein pair are ligated together in a traceless manner. Recently, a family of naturally split Inteins has been identified that is split at a noncanonical location in the primary sequence. These atypically split Inteins show considerable promise in protein engineering applications; however, the mechanism by which they associate is unclear and must be different from that of previously characterized canonically split Inteins due to unique topological restrictions. Here, we use a consensus design strategy to generate an atypical split Intein pair (Cat) that has greatly improved activity and is amenable to detailed biochemical and biophysical analysis. Guided by the solution structure of Cat, we show that the association of the fragments involves a disorder-to-order structural transition driven by hydrophobic interactions. This molecular recognition mechanism satisfies the top...
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an atypical mechanism of split Intein molecular recognition and folding
2018Co-Authors: Adam J. Stevens, Josef A. Gramespacher, David Cowburn, Giridhar Sekar, Tom W MuirAbstract:Split Inteins associate to trigger protein splicing in trans, a post-translational modification in which protein sequences fused to the Intein pair are ligated together in a traceless manner. Recently, a family of naturally split Inteins has been identified that is split at a noncanonical location in the primary sequence. These atypically split Inteins show considerable promise in protein engineering applications; however, the mechanism by which they associate is unclear and must be different from that of previously characterized canonically split Inteins due to unique topological restrictions. Here, we use a consensus design strategy to generate an atypical split Intein pair (Cat) that has greatly improved activity and is amenable to detailed biochemical and biophysical analysis. Guided by the solution structure of Cat, we show that the association of the fragments involves a disorder-to-order structural transition driven by hydrophobic interactions. This molecular recognition mechanism satisfies the topological constraints of the Intein fold and, importantly, ensures that premature chemistry does not occur prior to fragment complementation. Our data lead a common blueprint for split Intein complementation in which localized structural rearrangements are used to drive folding and regulate protein-splicing activity.
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Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis
2017Co-Authors: Josef A. Gramespacher, Adam J. Stevens, Duy P Nguyen, Jason W Chin, Tom W MuirAbstract:Naturally split Inteins have found widespread use in chemical biology due to their ability to drive the ligation of separately expressed polypeptides through a process termed protein trans-splicing (PTS). In this study, we harness PTS by rendering association of split Intein fragments conditional upon the presence of a user-defined protease. We show that these Intein “zymogens” can be used to create protein sensors and actuators that respond to the presence of various stimuli, including bacterial pathogens, viral infections, and light. We also show that this design strategy is compatible with several orthogonal split Intein pairs, thereby opening the way to the creation of multiplexed sensor systems.
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design of a split Intein with exceptional protein splicing activity
2016Co-Authors: Adam J. Stevens, David Cowburn, Neel H. Shah, Giridhar Sekar, Zachary Z Brown, Tom W MuirAbstract:Protein trans-splicing (PTS) by split Inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split Intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split Inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE Inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-Intein fusions. The du...
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Design of a Split Intein with Exceptional Protein Splicing Activity
2016Co-Authors: Adam J. Stevens, David Cowburn, Neel H. Shah, Zachary Z. Brown, Giridhar Sekar, Tom W MuirAbstract:Protein trans-splicing (PTS) by split Inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split Intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split Inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE Inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-Intein fusions. The durability and efficiency of Cfa should improve current Intein based technologies and may provide a platform for the development of new protein chemistry techniques
Xiang-qin Liu - One of the best experts on this subject based on the ideXlab platform.
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Engineered Ssp DnaX Inteins for protein splicing with flanking proline residues
2019Co-Authors: Xiao Zhang, Xiang-qin Liu, Qing MengAbstract:Inteins are internal protein sequences capable of catalyzing a protein splicing reaction by self-excising from a precursor protein and simultaneously joining the flanking sequences with a peptide bond. Split Inteins have separate pieces (N-Intein and C-Intein) that reassemble non-covalently to catalyze a protein trans-splicing reaction joining two polypeptides. Protein splicing has become increasingly useful tools in many fields of biological research and biotechnology. However, natural and engineered Inteins have failed previously to function when being flanked by proline residue at the −1 or +2 positions, which limits general uses of Inteins. In this study, different engineered Inteins were tested. We found that engineered Ssp DnaX mini-Intein and split Inteins could carry out protein splicing with proline at the +2 positions or at both −1 and +2 positions. Under in vivo conditions in E. coli cells, the mini-Intein, S1 split Intein, and S11 split Intein spliced efficiently, whereas the S0 split Intein did not splice with proline at both −1 and +2 positions. The S1 and S11 split Inteins also trans-spliced efficiently in vitro with proline at the +2 positions or at both −1 and +2 positions, but the S0 split Intein trans-spliced inefficiently with proline at the +2 position and did not trans-splice with proline at both −1 and +2 positions. These findings contribute significantly to the toolbox of Intein-based technologies by allowing the use of Inteins in proteins having proline at the splicing point. Keywords: Intein, Protein splicing, Flanking proline
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Amino acid sequences of mini-Inteins and split Inteins.
2013Co-Authors: Ying Lin, Qing Meng, Huiling Song, Xiang-qin LiuAbstract:Mini-Intein sequences are aligned using ClustalW online [26], and gaps (represented by -) were introduced to optimize the alignment. CneA PRP8 Intein was a natural mini-Intein. Ter DnaE-3 mini-Intein was derived from a natural conventional split Intein by a fusion of the Intein fragments. Other mini-Inteins were derived from natural Inteins by a deletion of their putative endonuclease domain sequences, with the position and number of deleted residues shown in parenthesis. A linker sequence (ASGHHHHHHGGSGS) was inserted at the site of deletion (or corresponding site in the CneA PRP8 and the Ter DnaE-3 mini-Inteins) and marked with an arrowhead. For each Intein, three (or two) amino acid residues (enclosed with a rectangle) of the native extein sequences on each side of the Intein were included in all splicing studies. In the Ssp DnaB mini-Intein, whose crystal structure is known, sequences of the 12 β-strands (β1 to β12) are underlined. Split sites for producing the S1 and S11 split Inteins are marked with black triangles.
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Protein Trans-Splicing of Multiple Atypical Split Inteins Engineered from Natural Inteins
2012Co-Authors: Ying Lin, Qing Meng, Huiling Song, Xiang-qin LiuAbstract:Protein trans-splicing by split Inteins has many uses in protein production and research. Splicing proteins with synthetic peptides, which employs atypical split Inteins, is particularly useful for site-specific protein modifications and labeling, because the synthetic peptide can be made to contain a variety of unnatural amino acids and chemical modifications. For this purpose, atypical split Inteins need to be engineered to have a small N-Intein or C-Intein fragment that can be more easily included in a synthetic peptide that also contains a small extein to be trans-spliced onto target proteins. Here we have successfully engineered multiple atypical split Inteins capable of protein trans-splicing, by modifying and testing more than a dozen natural Inteins. These included both S1 split Inteins having a very small (11–12 aa) N-Intein fragment and S11 split Inteins having a very small (6 aa) C-Intein fragment. Four of the new S1 and S11 split Inteins showed high efficiencies (85– 100%) of protein trans-splicing both in E. coli cells and in vitro. Under in vitro conditions, they exhibited reaction rate constants ranging from,1.761024 s21 to,3.861024 s21, which are comparable to or higher than those of previously reported atypical split Inteins. These findings should facilitate a more general use of trans-splicing between proteins and synthetic peptides, by expanding the availability of different atypical split Inteins. They also have implications on understanding the structure-function relationship of atypical split Inteins, particularly in terms of Intein fragment complementation
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highly efficient and more general cis and trans splicing Inteins through sequential directed evolution
2011Co-Authors: Julia H Applebytagoe, Henning D. Mootz, Ilka V. Thiel, Yi Wang, Yanfei Wang, Xiang-qin LiuAbstract:Inteins are internal protein sequences that post-translationally self-excise and splice together the flanking sequences, the so-called exteins. Natural and engineered Inteins have been used in many practical applications. However, Inteins are often inefficient or inactive when placed in a non-native host protein and may require the presence of several amino acid residues of the native exteins, which will then remain as a potential scar in the spliced protein. Thus, more general Inteins that overcome these limitations are highly desirable. Here we report sequential directed evolution as a new approach to produce Inteins with such properties. Random mutants of the Ssp (Synechocystis sp. PCC 6803) DnaB mini-Intein were inserted into the protein conferring kanamycin resistance at a site where the parent Intein was inactive for splicing. The mutants selected for splicing activity were further improved by iterating the procedure for two more cycles at different positions in the same protein. The resulting improved Inteins showed high activity in the positions of the first rounds of selection, in multiple new insertion sites, and in different proteins. One of these Inteins, the M86 mutant, which accumulated 8 amino acid substitutions, was also biochemically characterized in an artificially split form with a chemically synthesized N-terminal Intein fragment consisting of 11 amino acids. When compared with the unevolved split Intein, it exhibited an ∼60-fold increased rate in the protein trans-splicing reaction and a Kd value for the interaction of the split Intein fragments improved by an order of magnitude. Implications on the Intein structure-function, practical application, and evolution are discussed.
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novel split Intein for trans splicing synthetic peptide onto c terminus of protein
2009Co-Authors: Julia H Appleby, Gerrit Volkmann, Kaisong Zhou, Xiang-qin LiuAbstract:Conventional split Inteins have been useful for trans-splicing between recombinant proteins, and an artificial S1 split Intein is useful for adding synthetic peptide onto the N terminus of recombinant proteins. Here we have engineered a novel S11 split Intein for trans-splicing synthetic peptide onto the C terminus of recombinant proteins. The C-Intein of the S11 split Intein is extremely small (6 amino acids (aa)); thus it can easily be produced together with a synthetic C-extein to be added to the C terminus of target proteins. The S11 Intein was derived from the Ssp GyrB Intein after deleting the homing endonuclease domain and splitting the remaining Intein sequence near the C terminus, producing a 150-aa N-Intein (IN) and a 6-aa C-Intein (IC). Its trans-splicing activity was demonstrated first in Escherichia coli cells and then in vitro for trans-splicing between a synthetic peptide and a recombinant protein. The in vitro trans-splicing reaction exhibited a typical rate constant of (6.9+/-2.2)x10(-5) s(-1) and reached a high efficiency of approximately 80%. This S11 split Intein can be useful for adding any desirable chemical groups to the C terminus of a protein of interest, which may include modified and unnatural amino acids, biotin and fluorescent labels, and even drug molecules.
Kenneth V. Mills - One of the best experts on this subject based on the ideXlab platform.
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protein splicing activity of the haloferax volcanii polb c Intein is sensitive to homing endonuclease domain mutations
2020Co-Authors: Shachar Robinzon, Uri Gophna, Alexandra R Cawood, Mercedes Ruiz, Neta Altmanprice, Kenneth V. MillsAbstract:Inteins are selfish genetic elements residing in open reading frames that can splice post-translationally, resulting in the ligation of an uninterrupted, functional protein. Like other Inteins, the DNA polymerase B (PolB) Intein of the halophilic archaeon Haloferax volcanii has an active homing endonuclease (HEN) domain, facilitating its horizontal transmission. Previous work has shown that the presence of the PolB Intein exerts a significant fitness cost on the organism compared to an Intein-free isogenic H. volcanii. Here, we show that mutation of a conserved residue in the HEN domain not only reduces Intein homing but also slows growth. Surprisingly, although this mutation is far from the protein splicing active site, it also significantly reduces in vitro protein splicing. Moreover, two additional HEN domain mutations, which could not be introduced to H. volcanii, presumably due to lethality, also eliminate protein splicing activity in vitro. These results suggest an interplay between HEN residues and the protein splicing domain, despite an over 35 A separation in a PolB Intein homology model. The combination of in vivo and in vitro evidence strongly supports a model of codependence between the self-splicing domain and the HEN domain that has been alluded to by previous in vitro studies of protein splicing with HEN domain-containing Inteins.
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Recent advances in in vivo applications of Intein-mediated protein splicing
2014Co-Authors: Natalya I. Topilina, Kenneth V. MillsAbstract:Intein-mediated protein splicing has become an essential tool in modern biotechnology. Fundamental progress in the structure and catalytic strategies of cis- and trans- splicing Inteins has led to the development of modified Inteins that promote efficient protein purification, ligation, modification and cyclization. Recent work has extended these in vitro applications to the cell or to whole organisms. We review recent advances in Intein-mediated protein expression and modification, post-translational processing and labeling, protein regulation by conditional protein splicing, biosensors, and expression of trans -genes.
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Intramolecular Disulfide Bond between Catalytic Cysteines in an Intein Precursor
2012Co-Authors: Wen Chen, Kenneth V. Mills, Julie N. Reitter, Robert J. Linhardt, Jiajing Liu, Chunyu WangAbstract:Protein splicing is a self-catalyzed and spontaneous post-translational process in which Inteins excise themselves out of precursor proteins while the exteins are ligated together. We report the first discovery of an intramolecular disulfide bond between the two active-site cysteines, Cys1 and Cys+1, in an Intein precursor composed of the hyperthermophilic Pyrococcus abyssi PolII Intein and extein. The existence of this intramolecular disulfide bond is demonstrated by the effect of reducing agents on the precursor, mutagenesis, and liquid chromatography–mass spectrometry (LC–MS) with tandem MS (MS/MS) of the tryptic peptide containing the intramolecular disulfide bond. The disulfide bond inhibits protein splicing, and splicing can be induced by reducing agents such as tris(2-carboxyethyl)phosphine (TCEP). The stability of the intramolecular disulfide bond is enhanced by electrostatic interactions between the N- and C-exteins but is reduced by elevated temperature. The presence of this intramolecular disulfide bond may contribute to the redox control of splicing activity in hypoxia and at low temperature and point to the intriguing possibility that Inteins may act as switches to control extein function
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canonical protein splicing of a class 1 Intein that has a class 3 noncanonical sequence motif
2011Co-Authors: Julie N. Reitter, Kenneth V. MillsAbstract:A Thermobifida fusca Intein has two characteristics of class 3 Inteins: a noncontiguous covariant Trp-Cys-Thr triplet and a Ser flanking its C terminus. However, it has Cys at position one, characteristic of class 1 Inteins. Splicing does not require the internal Cys, which may instead coordinate the active site. Therefore, the Intein is class 1.
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Canonical Protein Splicing of a Class 1 Intein That Has a Class 3 Noncanonical Sequence Motif
2010Co-Authors: Julie N. Reitter, Kenneth V. MillsAbstract:A Thermobifida fusca Intein has two characteristics of class 3 Inteins: a noncontiguous covariant Trp-Cys-Thr triplet and a Ser flanking its C terminus. However, it has Cys at position one, characteristic of class 1 Inteins. Splicing does not require the internal Cys, which may instead coordinate the active site. Therefore, the Intein is class 1. Protein splicing is a posttranslational process by which an intervening polypeptide, or Intein, is responsible for its own excision from the flanking polypeptides, or exteins, concomi-tant with extein ligation (9, 10). The canonical mechanism of protein splicing has four steps (Fig. 1) (9, 10). First, the peptide bond linking the N-terminal extein (N extein) to the Intein is converted to a thioester or ester by nucleophilic attack by the side chain of the first Intein residue, Cys or Ser. Second, the first residue of the C-terminal extein (C extein) serves as a nucleophile to attack the nascent ester, resulting in transfer of the N extein from the side chai
Hannes M. Beyer - One of the best experts on this subject based on the ideXlab platform.
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mini Intein structures from extremophiles suggest a strategy for finding novel robust Inteins
2021Co-Authors: Mimmu Karoliina Hiltunen, Hannes M. Beyer, Hideo IwaiAbstract:Inteins are prevalent among extremophiles. Mini-Inteins with robust splicing properties are of particular interest for biotechnological applications due to their small size. However, biochemical and structural characterization has still been limited to a small number of Inteins, and only a few serve as widely used tools in protein engineering. We determined the crystal structure of a naturally occurring Pol-II mini-Intein from Pyrococcus horikoshii and compared all three mini-Inteins found in the genome of P. horikoshii. Despite their similar sizes, the comparison revealed distinct differences in the insertions and deletions, implying specific evolutionary pathways from distinct ancestral origins. Our studies suggest that sporadically distributed mini-Inteins might be more promising for further protein engineering applications than highly conserved mini-Inteins. Structural investigations of additional Inteins could guide the shortest path to finding novel robust mini-Inteins suitable for various protein engineering purposes.
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the convergence of the hedgehog Intein fold in different protein splicing mechanisms
2020Co-Authors: Hannes M. Beyer, Alexander Wlodawer, Kornelia M. Mikula, Salla I Virtanen, Sesilja A Aranko, George T Lountos, O Samuli H Ollila, Hideo IwaiAbstract: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.
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the crystal structure of the naturally split gp41 1 Intein guides the engineering of orthogonal split Inteins from cis splicing Inteins
2020Co-Authors: Hannes M. Beyer, Alexander Wlodawer, Kornelia M. Mikula, Hideo IwaiAbstract:Protein trans-splicing catalyzed by split Inteins has increasingly become useful as a protein engineering tool. We solved the 1.0 A-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.
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Crystal structures of CDC21-1 Inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site
2019Co-Authors: Hannes M. Beyer, Kornelia M. Mikula, Tatiana V. Kudling, Hideo IwaiAbstract: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.
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the crystal structure of the naturally split gp41 1 Intein guides the engineering of orthogonal split Inteins from a cis splicing Intein
2019Co-Authors: Hannes M. Beyer, Alexander Wlodawer, Kornelia M. Mikula, Hideo IwaiAbstract:Protein trans-splicing catalyzed by split Inteins has increasingly become useful as a protein engineering tool. The 1.0 A-resolution crystal structure of a variant from naturally split gp41-1 Intein, identified from the environmental metagenomic sequence data, revealed an improved pseudo-C2-symmetry commonly found in the Hedgehog/Intein (HINT) superfamily with extensive charge-charge interactions between the split N-and C-terminal Intein fragments. We successfully created orthogonal split Inteins by engineering a similar charge network in the same region of a cis-splicing Intein. The same strategy could be applicable for creating novel natural-like split Inteins from other, more prevalent cis-splicing Inteins.