Protein splicing of inteins with atypical glutamine and aspartate C-terminal residues

Inteins are protein-splicing domains present in many proteins. They self-catalyze their excision from the host protein, ligating their former flanks by a peptide bond. The C-terminal residue of inteins is typically an asparagine (Asn). Cyclization of this residue to succinimide causes the final detachment of inteins from their hosts. We studied protein-splicing activity of two inteins with atypical C-terminal residues. One having a C-terminal glutamine (Gln), isolated from Chilo iridescent virus (CIV), and another unique intein, first reported here, with a C-terminal aspartate, isolated from Carboxydothermus hydrogenoformans (Chy). Protein-splicing activity was examined in the wild-type inteins and in several mutants with N- and C-terminal amino acid substitutions. We demonstrate that both wild-type inteins can protein splice, probably by new variations of the typical protein-splicing mechanism. Substituting the atypical C-terminal residue to the typical Asn retained protein-splicing only in the CIV intein. All diverse C-terminal substitutions in the Chy intein (Asp(345) to Asn, Gln, Glu, and Ala) abolished protein-splicing and generated N- and C-terminal cleavage. The observed C-terminal cleavage in the Chy intein ending with Ala cannot be explained by cyclization of this residue. We present and discuss several new models for reactions in the protein-splicing pathway.     

Protein splicing

Protein splicing is a posttranslational process that results in excision of an internal protein region (intein) and ligation of its flanking sequences (exteins). As distinguished from other variants of protein processing, protein splicing does not require cofactors of enzymes. Protein splicing is catalyzed by an internal domain (so-called Hint domain) of the intein itself. The review considers the main regularities and molecular mechanisms of the process, as well as the functions of Hint domains in other protein families (Hh proteins, bacterial BIL domains, etc.). Studies of protein splicing are of importance from both theoretical and applied viewpoints. For instance, comparisons of the inteins found in different domains of life illustrate the role of horizontal transfer in intein spreading. A possible role of inteins in regulating several cell processes is discussed on the basis of recent data.     

Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments

Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases. In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected. The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids in which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E. coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively. One type of genetic alteration of the RecA intein involved deletion of the the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain. The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the N- and C-terminal segments of the intein, less than 110 aa each. Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region. The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans. The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each.     

NMR structure of a KlbA intein precursor from Methanococcus jannaschii

Certain proteins of unicellular organisms are translated as precursor polypeptides containing inteins (intervening proteins), which are domains capable of performing protein splicing. These domains, in conjunction with a single residue following the intein, catalyze their own excision from the surrounding protein (extein) in a multistep reaction involving the cleavage of two intein-extein peptide bonds and the formation of a new peptide bond that ligates the two exteins to yield the mature protein. We report here the solution NMR structure of a 186-residue precursor of the KlbA intein from Methanococcus jannaschii, comprising the intein together with N- and C-extein segments of 7 and 11 residues, respectively. The intein is shown to adopt a single, well-defined globular domain, representing a HINT (Hedgehog/Intein)-type topology. Fourteen beta-strands are arranged in a complex fold that includes four beta-hairpins and an antiparallel beta-ribbon, and there is one alpha-helix, which is packed against the beta-ribbon, and one turn of 3(10)-helix in the loop between the beta-strands 8 and 9. The two extein segments show increased disorder, and form only minimal nonbonding contacts with the intein domain. Structure-based mutation experiments resulted in a proposal for functional roles of individual residues in the intein catalytic mechanism.     

Trans protein splicing of cyanobacterial split inteins in endogenous and exogenous combinations

Inteins are autocatalytic protein domains that post-translationally excise from protein precursors and ligate their flanking regions with a peptide bond, in a process called protein splicing. Intein-containing DNA polymerases of cyanobacteria and nanoarchaea are naturally split into two separate genes at their intein domain. Such naturally occurring split inteins rapidly self-associate and reconstitute protein-splicing activity in trans. Here, we analyze the in vitro protein-splicing activity of three naturally split inteins from diverse cyanobacteria: Oscillatoria limnetica, Thermosynechococcus vulcanus, and Nostoc sp. PCC7120. N- and C-terminal halves of these split inteins were mixed in nine combinations, resulting in three endogenous (wild-type) and six exogenous combinations. Protein splicing was detected in all split-intein combinations, despite a 30-50% sequence variation between the homologous proteins. Splicing activity proceeded under a variety of conditions, including the presence of denaturants and reductants and high temperature, ionic strength, and viscosity. Still, in a high concentration of salt (2 M) or urea (6 M), specific combinations spliced significantly better than others. Additionally, copper ions were found to inhibit trans splicing in a reversible double-lock reaction. Our comparative analysis of naturally split inteins in endogenous and exogenous combinations demonstrates the modularity of trans protein-splicing elements and their robust activity. It suggests tight interactions between split-intein halves and conditions for modifying the specificity of intein parts. These results promote the biotechnological use of split inteins for controlled assembly of protein fragments either in vivo or in vitro and under moderate or extreme conditions.     

Distribution and function of new bacterial intein-like protein domains

Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein-splicing of their host-protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N-terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein-like (BIL) domains. Bacterial intein-like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C-terminal flank and could also protein-splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.     

Protein splicing

Inteins are internal polypeptide sequences that are posttranslationally excised from a protein precursor by a self-catalyzed protein-splicing reaction. Most of inteins consist of N- and C-terminal protein splicing domain and central endonuclease domain. The endonuclease domain can initiate mobility of the intein gene, this process being named intein homing. This review is focused on the recent data about the structure and function of inteins. Main intein-mediated protein-engineering applications, such as protein purification, ligation and cyclization, new forms of biosensors, are presented.     

Minimization and stabilization of the Mycobacterium tuberculosis recA intein

Many naturally occurring inteins consist of two functionally independent domains, a protein-splicing domain and an endonuclease domain. In a previous study, a 168 amino acid residue mini-intein was generated by removal of the central endonuclease domain of the 440 residue Mycobacterium tuberculosis (Mtu) recA intein. In addition, directed evolution experiments identified a mutation, V67L, that improved the activity of the mini-intein significantly. A recent crystal structure shows that the loop connecting two beta-strands from the N-terminal and C-terminal intein subdomains of the mini-intein is disordered. The goals of the present study were to generate smaller mini-intein derivatives and to understand the basis for reversal of the splicing defect by the V67L mutation. Guided by the structural information, we generated a number of derivatives 135 to 152 residues in length, with V67 or L67. All of the new minimal inteins are functional in splicing. In vivo selection experiments for function showed that by removal of the loop region, 137 residues may be the lower limit for full protein-splicing activity. In addition, the activation effect of the V67L mutation was observed to be universal for mini-inteins longer than 137 residues. Structural and functional analyses indicate that the role of the mutation is in stabilization of the mini-intein core.     

Modular organization of inteins and C-terminal autocatalytic domains.

Analysis of the conserved sequence features of inteins (protein "introns") reveals that they are composed of three distinct modular domains. The N-terminal (N) and C-terminal (C) domains are predicted to perform different parts of the autocatalytic protein splicing reaction. An optional endonuclease domain (EN) is shown to correspond to different types of homing endonucleases in different inteins. The N domain contains motifs predicted to catalyze the first steps of protein splicing, leading to the cleavage of the intein N terminus from its protein host. Intein N domain motifs are also found in C-terminal autocatalytic domains (CADs) present in hedgehog and other protein families. Specific residues in the N domain of intein and CADs are proposed to form a charge relay system involved in cleaving their N-termini. The intein C domain is apparently unique to inteins and contains motifs that catalyze the final protein splicing steps: ligation of the intein flanks and cleavage of its C terminus to release the free intein and spliced host protein. All intein EN domains known thus far have dodecapeptide (DOD, LAGLI-DADG) type homing endonuclease motifs. This work identifies an EN domain with an HNH homing-endonuclease motif and two new small inteins with no EN domains. One of these small inteins might be inactive or a "pseudo intein." The results suggest a modular architecture for inteins, clarify their origin and relationship to other protein families, and extend recent experimental findings on the functional roles of intein N, C, and EN motifs.     

Spontaneous proton transfer to a conserved intein residue determines on-pathway protein splicing

The discovery of inteins, which are protein-splicing elements, has stimulated interest for various applications in chemical biology, bioseparations, drug delivery, and sensor development. However, for inteins to effectively contribute to these applications, an increased mechanistic understanding of cleavage and splicing reactions is required. While the multistep chemical reaction that leads to splicing is often explored and utilized, it is not clear how the intein navigates through the reaction space. The sequence of reaction steps must progress in concert in order to yield efficient splicing while minimizing off-pathway cleavage reactions. In this study, we demonstrate that formation of a previously identified branched intermediate is the critical step for determining splicing over cleavage products. By combining experimental assays and quantum mechanical simulations, we identify the electrostatic interactions that are important to the dynamics of the reaction steps. We illustrate, via an animated simulation trajectory, a proton transfer from the first C-terminal extein residue to a conserved aspartate, which synchronizes the multistep enzymatic reaction that is key to splicing. This work provides new insights into the complex interplay between critical active-site residues in the protein splicing mechanism, thereby facilitating biotechnological application while shedding light on multistep enzyme activity.     

Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue

The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, DeltaI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (DeltaI-CM) increased C-terminal cleavage activity. Using the DeltaI-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, DeltaDeltaI(hh)-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue beta-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of DeltaI-SM, DeltaDeltaI(hh)-SM, and two variants, DeltaDeltaI(hh)-CM and DeltaDeltaI(hh), have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions.     

Protein splicing in the absence of an intein penultimate histidine

Protein splicing is a self-catalytic process in which an intervening sequence, termed an intein, is excised from a protein precursor, and the flanking polypeptides are religated. The conserved intein penultimate His facilitates this reaction by assisting in Asn cyclization, which results in C-terminal splice junction cleavage. However, many inteins do not have a penultimate His. Previous splicing studies with 2 such inteins yielded contradictory results. To resolve this issue, the splicing capacity of 2 more inteins without penultimate His residues was examined. Both the Methanococcus jannaschii phosphoenolpyruvate synthase and RNA polymerase subunit A' inteins spliced. Splicing of the phosphoenolpyruvate synthase intein improved when its penultimate Phe was changed to His, but splicing of the RNA polymerase subunit A' intein was inhibited when its penultimate Gly was changed to His. We propose that inteins lacking a penultimate His (i) arose by mutation from ancestors in which a penultimate His facilitated splicing, (ii) that loss of this His inhibited, but may not have blocked, splicing, and (iii) that selective pressure for efficient expression of the RNA polymerase yielded an intein that utilizes another residue to assist Asn cyclization, changing the intein active site so that a penultimate His now inhibits splicing.     

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

Protein splicing mediated by inteins is a self-processive reaction leading to the excision of the internal intein domain from a precursor protein and the concomitant ligation of the flanking sequences, the extein-N and extein-C parts, thereby reconstituting the host protein. Most inteins employ a splicing pathway in which the upstream scissile peptide bond is consecutively rearranged into two thioester or oxoester intermediates before intein excision and rearrangement into the new peptide bond occurs. The catalytically critical amino acids involved at the two splice junctions are cysteine, serine, or threonine. Notably, the only potential combination not observed so far in any of the known or engineered inteins corresponds to the transesterification from an oxoester to a thioester, which suggested that this formal uphill reaction with regard to the thermodynamic stability might be incompatible with intein-mediated catalysis. We show that corresponding mutations also led to inactive gp41-1 and AceL-TerL inteins. We report the novel GOS-TerL split intein identified from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 residues. Mutational analysis showed that its efficient splicing reaction indeed follows the shift from oxoester to thioester and thus represents a rare diversion from the canonical pathway. Furthermore, the GOS-TerL intein has an atypical split site close to the N terminus. The Int^N fragment could be shortened from 37 to 28 amino acids and exchanged with the 25-amino acid Int^N fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the Int^C fragment of the GOS-TerL intein.     

Protein splicing of the three Pyrococcus abyssi ribonucleotide reductase inteins

An intein is a polypeptide that interrupts the functional domains of a protein, called the exteins. The intein can facilitate its own excision from the exteins, concomitant with the ligation of the exteins, in a process called protein splicing. The alpha subunit of the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi is interrupted by three inteins in separate insertion sites. Each intein can facilitate protein splicing when over-expressed in Escherichia coli, with affinity domains serving as the exteins. The influence of the N-terminal flanking residue on the efficiency of splicing is specific to each intein. Each intein has a different downstream nucleophilic residue, and cannot tolerate substitution to a residue of lesser or equal nucleophilicity. The influence of the conserved penultimate His also differs between the inteins.     

蛋白质剪接研究进展

蛋白质剪接是一个翻译后自催化加工过程,它不需要酶或其他辅助因子的参与。在这个过程中,前体蛋白的Intein(内含肽)被切离,其两侧的Extein(外显肽)连接在一起。Intein按结构可分为经典Intein和微型Intein,其中的经典Intein包括Hint结构域和中间的归巢内切酶结构域(该结构域在微型内含肽中不存在)。蛋白质剪接及其他具有Hint结构域的蛋白加工过程的起始步骤是N-S/O酰基重排反应,该反应是由Hint结构域催化的;Intein的剪接还分为顺式剪接和反式剪接,通过对Intein进行改造,可以阻断剪接过程,但不影响N端肽键或C端肽键的断裂;通过筛选突变体,可以获得温度敏感型、pH敏感型或小分子诱导型的内含肽。这些研究促进了Intein在多肽制备及其它方面的应用。     

Structural and mutational studies of a hyperthermophilic intein from DNA polymerase II of Pyrococcus abyssi

Protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). Protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. We report the solution NMR structures of the hyperthermophilic Pyrococcus abyssi PolII intein, which has a noncanonical C-terminal glutamine instead of an asparagine. The NMR structures were determined to a backbone root mean square deviation of 0.46 ? and a heavy atom root mean square deviation of 0.93 ?. The Pab PolII intein has a common HINT (hedgehog intein) fold but contains an extra β-hairpin that is unique in the structures of thermophilic inteins. The NMR structures also show that the Pab PolII intein has a long and disordered loop in place of an endonuclease domain. The N-terminal Cys-1 amide is hydrogen bonded to the Thr-90 hydroxyl in the conserved block-B TXXH motif and the Cys-1 thiol forms a hydrogen bond with the block F Ser-166. Mutating Thr-90 to Ala dramatically slows N-terminal cleavage, supporting its pivotal role in promoting the N-S acyl shift. Mutagenesis also showed that Thr-90 and His-93 are synergistic in catalyzing the N-S acyl shift. The block F Ser-166 plays an important role in coordinating the steps of protein splicing. NMR spin relaxation indicates that the Pab PolII intein is significantly more rigid than mesophilic inteins, which may contribute to the higher optimal temperature for protein splicing.     

Protein splicing of a Pyrococcus abyssi intein with a C-terminal glutamine

Protein splicing involves the excision of an intervening polypeptide sequence, the intein, from a precursor protein and the concomitant ligation of the flanking polypeptides, the exteins, by a peptide bond. Most reported inteins have a C-terminal asparagine residue, and it has been shown that cyclization of this residue is coupled to peptide bond cleavage between the intein and C-extein. We show that the intein interrupting the DNA polymerase II DP2 subunit in Pyrococcus abyssi, which has a C-terminal glutamine, is capable of facilitating protein splicing. Substitution of an asparagine for the C-terminal glutamine moderately improves the rate and extent of protein splicing. However, substitution of an alanine for the penultimate histidine residue, with either asparagine or glutamine in the C-terminal position, prevents protein splicing and facilitates cleavage at the intein N terminus. The intein facilitates in vitro protein splicing only at temperatures above 30 degrees C and can be purified as a nonspliced precursor. This temperature dependence has enabled us to characterize the optimal in vitro splicing conditions and determine the rate constants for splicing as a function of temperature.     

Mutational analysis of protein splicing, cleavage, and self-association reactions mediated by the naturally split Ssp DnaE intein

The ability to separately purify the naturally split Synechocystis sp. PCC6803 (Ssp) DnaE intein domains has allowed detailed examination of both universal and Ssp DnaE intein-specific steps in the protein splicing pathway. By engineering substitutions at both the +1 and penultimate intein positions, we have further characterized intein reaction kinetics in this system. Replacement of the crucial +1Cys with serine decreased N-terminal cleavage and trans-splicing rates; however, this substitution did not prevent splicing or the ability of ZnCl2 to inhibit it. Substitution of the penultimate intein residue (alanine) with a typically conserved histidine did not increase the rate or extent of trans-splicing or cleavage under typical assay conditions. Despite the observation that this histidine aids in asparagine cyclization for other inteins, it did not encourage C-terminal cleavage for the Ssp DnaE intein or uncouple it from N-terminal cleavage. Both the +1Ser and Ala to His mutants were insensitive to ZnCl2 during trans-cleavage experiments, uncoupling a previously linked inhibition in asparagine cyclization from an inhibition in trans-thioesterification detected for the wild-type intein.     

Expanding the definition of class 3 inteins and their proposed phage origin

Inteins are the protein equivalent of introns. Their protein splicing activity is essential for the host protein's maturation and function. Inteins are grouped into three classes based on sequence signature and splicing mechanism. The sequence signature of the recently characterized class 3 inteins is a noncontiguous Trp-Cys-Thr (WCT) motif and the absence of the standard class 1 Cys1 or Ser1 N-terminal nucleophile. The intein N-terminal Cys1 or Ser1 residue is essential for splicing in class 1 inteins. The mycobacteriophage Catera Gp206, Nocardioides sp. strain JS614 TOPRIM, and Thermobifida fusca YX Tfu2914 inteins have a mixture of class 1 and class 3 motifs. They carry the class 3 Trp-Cys-Thr motif and have the standard class 1 N-terminal Ser1 or Cys1. 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 Ser1) is a class 3 intein, and its Ser1 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.     

Protein splicing mechanisms and applications

Inteins are protein splicing elements that employ standard enzyme strategies to excise themselves from precursor proteins and ligate the surrounding sequences (exteins). The protein splicing pathway consists of four nucleophilic displacements directed by the intein plus the first C-extein residue. The intein active site(s) are formed by folding of the intein within the precursor, which brings together the splice junctions and internal intein residues that assist catalysis. Inteins with non-canonical catalytic residues splice by modified pathways. Understanding intein proteolytic cleavage and ligation activities has led to the development of many novel applications in the fields of protein engineering, enzymology, microarray production, target detection and activation of transgenes in plants. Recent advances include intein-mediated attachment of proteins to solid supports for microarray or western blot analysis, linking nucleic acids to proteins and controllable splicing, which converts inteins into molecular switches.