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.     

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。     

Creation of an artificial bifunctional intein by grafting a homing endonuclease into a mini-intein

The majority of inteins are comprised of a protein splicing domain and a homing endonuclease domain. Experimental evidence has demonstrated that the splicing domain and the endonuclease domain in a bifunctional intein are largely independent of each other with respect to both structure and activity. Here, an artificial bifunctional intein has been created through the insertion of an existing homing endonuclease into a mini-intein that is naturally lacking this functionality. The gene for I-CreI, an intron-encoded homing endonuclease, was grafted into the monofunctional Mycobacterium xenopi GyrA intein at the putative site of the missing endonuclease. The resulting fusion protein was found to be capable of protein splicing similar to that of the parent intein. In addition, the protein demonstrated site-specific endonuclease activity that is characteristic of the I-CreI homing endonuclease. The function of each domain therefore remained unaffected by the presence of the other domain. This artificial fusion of the two domains is a potential novel mobile genetic element.     

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.     

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.     

The Mycobacterium xenopi GyrA protein splicing element: characterization of a minimal intein.

The 198-amino-acid in-frame insertion in the gyrA gene of Mycobacterium xenopi is the smallest known naturally occurring active protein splicing element (intein). Comparison with other mycobacterial gyrA inteins suggests that the M. xenopi intein underwent a complex series of events including (i) removal of 222 amino acids that encompass most of the central intein domain, and (ii) addition of a linker of unrelated residues. This naturally occurring genetic rearrangement is a representative characteristic of the taxon. The deletion process removes the conserved motifs involved in homing endonuclease activity. The linker insertion represents a structural requirement, as its mutation resulted in failure to splice. The M. xenopi GyrA intein thus provides a paradigm for a minimal protein splicing element.     

Protein splicing and auto-cleavage of bacterial intein-like domains lacking a C'-flanking nucleophilic residue

Bacterial intein-like (BIL) domains are newly identified homologs of intein protein-splicing domains. The two known types of BIL domains together with inteins and hedgehog (Hog) auto-processing domains form the Hog/intein (HINT) superfamily. BIL domains are distinct from inteins and Hogs in sequence, phylogenetic distribution, and host protein type, but little is known about their biochemical activity. Here we experimentally study the auto-processing activity of four BIL domains. An A-type BIL domain from Clostridium thermocellum showed both protein-splicing and auto-cleavage activities. The splicing is notable, because this domain has a native Ala C'-flanking residue rather than a nucleophilic residue, which is absolutely necessary for intein protein splicing. B-type BIL domains from Rhodobacter sphaeroides and Rhodobacter capsulatus cleaved their N' or C' ends. We propose an alternative protein-splicing mechanism for the A-type BIL domains. After an initial N-S acyl shift, creating a thioester bond at the N' end of the domain, the C' end of the domain is cleaved by Asn cyclization. The resulting amino end of the C'-flank attacks the thioester bond next at the N' end of the domain. This aminolysis step splices the two flanks of the domain. The B-type BIL domain cleavage activity is explained in the context of the canonical intein protein-splicing mechanism. Our results suggest that the different HINT domains have related biochemical activities of proteolytic cleavages, ligation and splicing. Yet the predominant reactions diverged in each HINT type according to their specific biological roles. We suggest that the BIL domain cleavage and splicing reactions are mechanisms for post-translationally generating protein variability, particularly in extracellular bacterial proteins.     

Split dnaE genes encoding multiple novel inteins in Trichodesmium erythraeum

Three inteins were found when analyzing a pair of split dnaE genes encoding the catalytic subunit of DNA polymerase III in the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. The three inteins (DnaE-1, DnaE-2, and DnaE-3) were clustered in a 70-amino acid (aa) region of the predicted DnaE protein. The DnaE-1 intein is 1258 aa long and three times as large as a typical intein, due to the presence of large tandem repeats in which a 57-aa sequence is repeated 17 times. The DnaE-2 intein has a more typical size of 428 aa with putative protein splicing and endonuclease domains. The DnaE-3 intein is a split intein consisting of a 102-aa N-terminal part and a 36-aa C-terminal part encoded on the first and second split dnaE genes, respectively. Synthesis of a mature DnaE protein is predicted to involve expression of two split dnaE genes followed by two protein cis-splicing reactions and one protein trans-splicing reaction. Tandem repeats in the DnaE-1 intein inhibited the protein splicing activity of this intein when tested in Escherichia coli cells and may potentially regulate DnaE synthesis in vivo.     

Characterisation of the coccolithovirus intein

The identification of inteins in viral genomes is becoming increasingly common. Inteins are selfish DNA elements found within coding regions of host proteins. Following translation, they catalyse their own excision and the formation of a peptide bond between the flanking protein regions. Many inteins also display homing endonuclease function. Here, the newly identified coccolithovirus intein is described and is predicted to have both self-splicing and homing endonuclease activity. The biochemical mechanism of its protein splicing activity is hypothesised, and the prevalence of the intein among natural coccolithovirus isolates is tested.     

Characterization of a naturally occurring trans-splicing intein from Synechocystis sp. PCC6803

A naturally occurring trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) was used to characterize the intein-catalyzed splicing reaction. Trans-splicing/cleavage reactions were initiated by combining the N-terminal splicing domain of the Ssp DnaE intein containing five native N-extein residues and maltose binding protein as the N-extein with the C-terminal Ssp DnaE intein splicing domain (E(C)) with or without thioredoxin fused in-frame to its carboxy terminus. Observed rate constants (k(obs)) for dithiothreitol-induced N-terminal cleavage, C-terminal cleavage, and trans-splicing were (1.0 +/- 0.5) x 10(-3), (1.9 +/- 0.9) x 10(-4), and (6.6 +/- 1.3) x 10(-5) s(-1), respectively. Preincubation of the intein fragments showed no change in k(obs), indicating association of the two splicing domains is rapid relative to the subsequent steps. Interestingly, when E(C) concentrations were substoichiometric with respect to the N-terminal splicing domain, the levels of N-terminal cleavage were equivalent to the amount of E(C), even over a 24 h period. Activation energies for N-terminal cleavage and trans-splicing were determined by Arrhenius plots to be 12.5 and 8.9 kcal/mol, respectively. Trans-splicing occurred maximally at pH 7.0, while a slight increase in the extent of N-terminal cleavage was observed at higher pH values. This work describes an in-depth kinetic analysis of the splicing and cleavage activity of an intein, and provides insight for the use of the split intein as an affinity domain.     

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.     

断裂蛋白质内含子的剪接机制、起源和进化

蛋白质内含子(intein)是具有自我催化活性的蛋白质. 翻译后,通过蛋白质剪接从蛋白质前体中去掉,并以肽键连接两侧蛋白质外显子(extein)形成成熟蛋白质. 断裂蛋白质内含子(split intein)在蛋白质内含子中部区域特定位点发生断裂,形成N端片段和C端片段,分别由基因组上相距较远的两个基因编码. 现在已知,它仅分布于蓝细菌和古细菌中. 断裂蛋白质内含子的N端片段和C端片段通过非共价键(如静电作用)相互识别,重建催化活性中心,介导蛋白质反式剪接. 断裂蛋白质内含子的发现进一步深化了人们对基因表达和蛋白质翻译后成熟过程复杂性的认识,而且它在蛋白质工程、蛋白质药物开发和蛋白质结构与功能研究等方面有非常广泛的应用. 本文试图综述断裂蛋白质内含子的分布、结构特征和剪接机制,并分析其可能的起源和进化途径.     

The nuclear-encoded inteins of fungi

An intein is a protein sequence embedded within a precursor protein that is excised during protein maturation. Inteins were first found encoded in the VMA gene of Saccharomyces cerevisiae. Subsequently, they have been found in diverse organisms (eukaryotes, archaea, eubacteria and viruses). The VMA intein has been found in various saccharomycete yeasts but not in other fungi. Different inteins have now been found widely in the fungi (ascomycetes, basidiomycetes, zygomycetes and chytrids) and in diverse proteins. A protein distantly related to inteins, but closely related to metazoan hedgehog proteins, has been described from Glomeromycota. Many of the newly described inteins contain homing endonucleases and some of these are apparently active. The enlarged fungal intein data set permits insight into the evolution of inteins, including the role of horizontal transfer in their persistence. The diverse fungal inteins provide a resource for biotechnology using their protein splicing or homing endonuclease capabilities.     

Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources

Inteins excise themselves out of precursor proteins by the protein splicing reaction and have emerged as valuable protein engineering tools in numerous and diverse biotechnological applications. Split inteins have recently attracted particular interest because of the opportunities associated with generating a protein from two separate polypeptides and with trans-cleavage applications made possible by split intein mutants. However, natural split inteins are rare and differ greatly in their usefulness with regard to the achievable rates and yields. Here we report the first functional characterization of new split inteins previously identified by bioinformatics from metagenomic sources. The N- and C-terminal fragments of the four inteins gp41-1, gp41-8, NrdJ-1, and IMPDH-1 were prepared as fusion constructs with model proteins. Upon incubation of complementary pairs, we observed trans-splicing reactions with unprecedented rates and yields for all four inteins. Furthermore, no side reactions were detectable, and the precursor constructs were consumed virtually quantitatively. The rate for the gp41-1 intein, the most active intein on all accounts, was k = 1.8 ± 0.5 × 10(-1) s(-1), which is ～10-fold faster than the rate reported for the Npu DnaE intein and gives rise to completed reactions within 20-30 s. No cross-reactivity in exogenous combinations was observed. Using C1A mutants, all inteins were efficient in the C-terminal cleavage reaction, albeit at lower rates. C-terminal cleavage could be performed under a wide range of reaction conditions and also in the absence of native extein residues flanking the intein. Thus, these inteins hold great potential for splicing and cleavage applications.     

蛋白质剪切及其应用

蛋白质剪切是一种翻译后修饰事件 ,它将插入前体蛋白的中间的蛋白质肽段 (Intein ,internalproteinfrag ment)剪切出来 ,并用正常肽键将两侧蛋白质多肽链 (Extein ,flankingproteinfragments)连接起来。在此过程中不需要辅酶或辅助因子的作用 ,仅需四步分子内反应。Intein及其侧翼序列可以通过突变产生高度特异性的自我切割用于蛋白质纯化、蛋白质连接和蛋白质环化反应 ,在蛋白质工程方面有广泛的应用前景。     

蛋白内含子与蛋白剪接

蛋白内含子和蛋白剪接是蛋白质研究的前沿领域。重点介绍了蛋白内含子的结构和蛋白剪接机理的最新研究成果 ;蛋白内含子如同RNA剪接中的内含子 ,也是一类可移动的遗传元件 ;蛋白内含子目前研究的热点是蛋白内含子的功能研究及其在蛋白质工程和其它生物工程领域的用。     

Crystal structure of an archaeal intein-encoded homing endonuclease PI-PfuI

Inteins possess two different enzymatic activities, self-catalyzed protein splicing and site-specific DNA cleavage. These endonucleases, which are classified as part of the homing endonuclease family, initiate the mobility of their genetic elements into homologous alleles. They recognize long asymmetric nucleotide sequences and cleave both DNA strands in a monomer form. We present here the 2.1 A crystal structure of the archaeal PI-PfuI intein from Pyroccocus furiosus. The structure reveals a unique domain, designated here as the Stirrup domain, which is inserted between the Hint domain and an endonuclease domain. The horseshoe-shaped Hint domain contains a catalytic center for protein splicing, which involves both N and C-terminal residues. The endonuclease domain, which is inserted into the Hint domain, consists of two copies of substructure related by an internal pseudo 2-fold axis. In contrast with the I-CreI homing endonuclease, PI-PfuI possibly has two asymmetric catalytic sites at the center of a putative DNA-binding cleft formed by a pair of four-stranded beta-sheets. DNase I footprinting experiments showed that PI-PfuI covers more than 30 bp of the substrate asymmetrically across the cleavage site. A docking model of the DNA-enzyme complex suggests that the endonuclease domain covers the 20 bp DNA duplex encompassing the cleavage site, whereas the Stirrup domain could make an additional contact with another upstream 10 bp region. For the double-strand break, the two strands in the DNA duplex were cleaved by PI-PfuI with different efficiencies. We suggest that the cleavage of each strand is catalyzed by each of the two non-equivalent active sites.     

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.     

A topA intein in Pyrococcus furiosus and its relatedness to the r-gyr intein of Methanococcus jannaschii

A new intein coding sequence was found in a topA (DNA topoisomerase I) gene by cloning and sequencing this gene from the hyperthermophilic Archaeon Pyrococcus furiosus. The predicted Pfu topA intein sequence is 373 amino acids long and located two residues away from the catalytic tyrosine of the topoisomerase. It contains putative intein sequence blocks (C, E, and H) associated with intein endonuclease activity, in addition to intein sequence blocks (A, B, F, and G) that are necessary for protein splicing. This DNA topoisomerase I intein is most related to a reverse gyrase intein from the methanogenic Archaeon Methanococcus jannaschii. These two inteins share 31% amino acid sequence identity and, more importantly, have the same insertion sites in their respective host proteins. It is suggested that these two inteins are homologous inteins present in structurally related, but functionally distinct, proteins, with implications on intein evolution and intein homing.