Structural and Dynamical Features of Inteins and Implications on Protein Splicing

Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoe-like pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of intein-based technologies.     

Cisplatin inhibits protein splicing, suggesting inteins as therapeutic targets in mycobacteria

Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC(50) of ～2 μM, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ～40 μM, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria.     

Distribution of split DnaE inteins in cyanobacteria

Inteins are genetic elements found inside the coding regions of different host proteins and are translated in frame with them. The intein-encoded protein region is removed by an autocatalytic protein-splicing reaction that ligates the host protein flanks with a peptide bond. This reaction can also occur in trans with the intein and host protein split in two. After translation of the two genes, the two intein parts ligate their flanking protein parts to each other, producing the mature protein. Naturally split inteins are only known in the DNA polymerase III alpha subunit (polC or dnaE gene) of a few cyanobacteria. Analysing the phylogenetic distribution and probable genetic propagation mode of these split inteins, we conclude that they are genetically fixed in several large cyanobacterial lineages. To test our hypothesis, we sequenced parts of the dnaE genes from five diverse cyanobacteria and found all species to have the same type of split intein. Our results suggest the occurrence of a genetic rearrangement in the ancestor of a large division of cyanobacteria. This event fixed the dnaE gene in a unique two-genes one-protein configuration in the progenitor of many cyanobacteria. Our hypothesis, findings and the cloning procedure that we established allow the identification and acquisition of many naturally split inteins. Having a large and diverse repertoire of these unique inteins will enable studies of their distinct activity and enhance their use in biotechnology.     

Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution

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 K(d) 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.     

Splicing of the Mycobacteriophage Bethlehem DnaB Intein: IDENTIFICATION OF A NEW MECHANISTIC CLASS OF INTEINS THAT CONTAIN AN OBLIGATE BLOCK F NUCLEOPHILE*?

Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala¹ inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.     

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.     

定向进化提高微小蛋白质内含子Ter DnaE-3 剪接活性的研究

目前,蛋白质内含子在蛋白质工程领域中得到越来越广泛的应用。为提高微小蛋白质内含子Ter DnaE-3(Trichodesmium erythraeum)在异源宿主中的剪接活性,采用易错PCR技术,通过改变反应体系中dNTP、Mg2+、Mn2+的浓度等手段,借助依赖卡那霉素的蛋白质内含子筛选系统进行筛选。Western印迹结果表明:通过定向进化,其中5号突变体的剪接活性从原始的约20%提高至约85%;9号突变体能够避免发生剪接副反应,即N端断裂反应。氨基酸突变位点与剪接活性变化的相关性分析表明:参与α-helix形成的氨基酸的突变极有可能影响蛋白质内含子的断裂反应,参与β-sheet形成的氨基酸的突变则有可能影响蛋白质内含子结构的紧凑性。通过定向进化提高微小蛋白质内含子Ter DnaE-3在异源宿主中的剪接活性,进一步验证依赖卡那霉素抗性的筛选系统的可行性,为扩大蛋白质内含子的应用范围奠定基础。     

PRP8 Intein in Onygenales: Distribution and Phylogenetic Aspects

Inteins (internal proteins) are mobile genetic elements, inserted in housekeeping proteins, with self-splicing properties. Some of these elements have been recently pointed out as modulators of genetic expression or protein function. Herein, we evaluated, in silico, the distribution and phylogenetic patterns of PRP8 intein among 93 fungal strains of the order Onygenales. PRP8 intein(s) are present in most of the species (45/49), mainly as full-length inteins (containing both the Splicing and the Homing Endonuclease domains), and must have transferred vertically in all lineages, since their phylogeny reflects the group phylogeny. While the distribution of PRP8 intein(s) varies among species of Onygenaceae family, being absent in Coccidioides spp. and present as full and mini-intein in other species, they are consistently observed as full-length inteins in all evaluated pathogenic species of the Arthrodermataceae and Ajellomycetaceae families. This conservative and massive PRP8 intein presence in Ajellomycetacean and Arthrodermatecean species reinforces the previous idea that such genetic elements do not decrease the fungal fitness significantly and even might play some role in the host–pathogen relationship, at least in these two fungal groups. We may better position the species Ophidiomyces ophiodiicola (with no intein) in the Onygenaceae family and Onygena corvina (with a full-length intein) as a basal member in the Arthrodermataceae family.

     

Protein Splicing: How Inteins Escape from Precursor Proteins

Inteins are nature''s escape artists; they facilitate their excision from flanking polypeptides (exteins) concomitant with extein ligation to produce a mature host protein. Splicing requires sequential nucleophilic displacement reactions catalyzed by strategies similar to proteases and asparagine lyases. Inteins require precise reaction coordination rather than rapid turnover or tight substrate binding because they are single turnover enzymes with covalently linked substrates. This has allowed inteins to explore alternative mechanisms with different steps or to use different methods for activation and coordination of the steps. Pressing issues include understanding the underlying details of catalysis and how the splicing steps are controlled.     

PRP8 inteins in species of the genus Botrytis and other ascomycetes

The mobile elements termed inteins have a sporadic distribution in microorganisms. It is unclear how these elements are maintained. Inteins are intervening protein sequences that autocatalytically excise themselves from a precursor. Excision is a post-translational process referred to as 'protein splicing' in which the sequences flanking the intein are ligated, reforming the mature host protein. Some inteins contain a homing endonuclease domain (HEG) that is proposed to facilitate propagation of the intein element within a gene pool. We have previously demonstrated that the HEG of the PRP8 intein is highly active during meiosis in Botrytis cinerea. Here we analysed the Prp8 gene status in 21 additional Botrytis species to obtain insight into the mode of intein inheritance within the Botrytis lineage. Of the 21 species, 15 contained a PRP8 intein whereas six did not. The analysis was extended to closely related (Sclerotiniaceae) and distantly related (Ascomycota) taxa, focussing on evolutionary diversification of the PRP8 intein, including their possible acquisition by horizontal transfer and loss by deletion. Evidence was obtained for the occurrence of genetic footprints of previous intein occupation. There is no compelling evidence of horizontal transfer among species. Three distinct states of the Prp8 allele were identified, distributed over different orders within the Ascomycota: an occupied allele; an empty allele that was never occupied; an empty allele that was presumably previously occupied, from which the intein was precisely deleted. The presence of the genetic footprint identifies 20 species (including Neurospora crassa, Magnaporthe oryzae and Fusarium oxysporum) that previously contained the intein but have lost it entirely, while only 18 species (including Podospora anserina and Fusarium graminearum) appear never to have contained a PRP8 intein. The analysis indicates that inteins may be maintained in an equilibrium state.     

Engineered Ssp DnaX inteins for protein splicing with flanking proline residues

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.     

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.     

Intein spread and extinction in evolution

Inteins are selfish DNA elements found within coding regions. They are translated with their host protein, but then catalyze their own excision and the formation of a peptide bond between their flanking protein regions. Understanding what drives and selects inteins is relevant for assessing whether they have unidentified biological functions and whether they can invade and become established in new genes and organisms. Inteins are suggested to have been present and more common in the progenitors of eukaryotes and prokaryotes. In these cells, inteins had some beneficial function or had evolved from an unknown beneficial protein. Since then, this putative benefit has been lost and inteins are gradually becoming extinct. The proteins in which inteins are currently found are proposed to be proteins vital for the survival of the organism, where intein removal is most difficult.     

Kinetic and spectroscopic characterization of native and metal-substituted beta-lactamase from Aeromonas hydrophila AE036

Five new inteins were discovered in a survey of 39 mycobacterial strains that was undertaken to clarify the role of RecA inteins in mycobacteria. They are all inserted at the RecA-b site of the recA gene of Mycobacterium chitae, M. fallax, M. gastri, M. shimodei and M. thermoresistibile and belong to the MleRecA allelic family. Sequence analysis showed that although only M. tuberculosis harbours an intein at the RecA-a site the sequence of the RecA-b site is well conserved between species. Furthermore, the presence of inteins does not correlate with specific characteristics of the species such as pathogenicity or growth rate.     

The ins and outs of protein splicing elements

Protein splicing involves the removal of an internal protein sequence from a precursor molecule and the ligation of the two flanking sequences to produce a mature protein product, in a post-translational event analogous to the removal of an intron from rRNA. Protein splicing introns, or‘inteins’appear to be a novel type of genetic element capable of mediating gene conversion of an‘intein-less’allele, and hence promoting their own dissemination. The mechanism by which protein splicing is achieved is probably entirety encoded within the internal protein sequence, or intein, and does not require other accessory molecules. Although the concept of protein splicing inteins as selfish genetic elements of no immediate consequence to the host organism has emerged, this interpretation is questioned by recent evidence that in at least one example there appears to have been selection for protein splicing.     

Minimization of a eukaryotic mini-intein

Inteins are internal protein splicing elements that can autocatalytically self-excise from their host protein and ligate the protein flanks (exteins) with a peptide bond. Large inteins comprise independent protein splicing and endonuclease domains whereas mini-inteins lack the central endonuclease domain. To identify mini-intein domains that are essential for protein splicing, deletions were introduced at different sites of the 157-aa PRP8 mini-intein of Penicillium chrysogenum. The removal of eight and six amino acids at two different sites resulted in a functional eukaryotic mini-intein of only 143 aa.     

SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein histidines

Inteins are auto-processing domains that implement a multistep biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe–S] cluster assembly protein SufB from Mycobacterium tuberculosis (Mtu). Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wildtype (WT) precursor protein. Structural analysis and molecular dynamics (MD) simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N–S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen-specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.     

Invasion of a multitude of genetic niches by mobile endonuclease genes

Persistence of a mobile DNA element in a population reflects a balance between the ability of the host to eliminate the element and the ability of the element to survive and to disseminate to other individuals. In each of the three biological kingdoms, several families of a mobile DNA element have been identified which encode a single protein that acts on nucleic acids. Collectively termed homing endonuclease genes (HEGs), these elements employ varied strategies to ensure their survival. Some members of the HEG families have a minimal impact on host fitness because they associate with genes having self-splicing introns or inteins that remove the HEGs at the RNA or protein level. The HEG and the intron/intein gene spread throughout the population by a gene conversion process initiated by the HEG-encoded endonuclease called 'homing' in which the HEG and intron/intein genes are copied to cognate alleles that lack them. The endonuclease activity also contributes to a high frequency of lateral transmission of HEGs between species as has been documented in plants and other systems. Other HEGs have positive selection value because the proteins have evolved activities that benefit their host organisms. The success of HEGs in colonizing diverse genetic niches results from the flexibility of the encoded endonucleases in adopting new specificities.     

Trans-splicing of an artificially split fungal mini-intein

Inteins are internal protein domains found inside the coding region of different proteins. They can autocatalytically self-excise from their host protein and ligate the protein flanks, called exteins, with a peptide bond via a post-translational process called protein cis-splicing. In contrast, protein trans-splicing involves inteins split into an N- and a C-terminal domain. Both domains are synthesized as two separate components and each joined to an extein; the intein domains can reassemble and link the joined exteins into one functional protein. In this study, we introduced three split sites into the PRP8 mini-intein of Penicillium chrysogenum and demonstrated for the first time trans-splicing of a fungal PRP8 intein. Two of the sites introduced allowed splicing to occur in trans while the third was not functional.