Selection and Structure of Hyperactive Inteins: Peripheral Changes Relayed to the Catalytic Center

Inteins are phylogenetically diverse self-splicing proteins that are of great functional, evolutionary, biotechnological, and medical interest. To address the relationship between intein structure and function, particularly with respect to regulating the splicing reaction, and to groom inteins for application, we developed a phage display system to extend current in vivo selection for enhanced intein function to selection in vitro. We thereby isolated inteins that can function under excursions in temperature, pH, and denaturing environment. Remarkably, most mutations mapped to the surface of the intein, remote from the active site. We chose two mutants with enhanced splicing activity for crystallography, one of which was also subjected to NMR analysis. These studies define a “ripple effect”, whereby mutations in peripheral non-catalytic residues can cause subtle allosteric changes in the active-site environment in a way that facilitates intein activity. Altered salt-bridge formation and chemical shift changes of the mutant inteins provide a molecular rationale for their phenotypes. These fundamental insights will advance the utility of inteins in chemical biology, biotechnology, and medicine.     

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.     

Backbone Dynamics and Global Effects of an Activating Mutation in Minimized Mtu RecA Inteins

Inteins mediate protein splicing, which has found many applications in biotechnology and protein engineering. A single valine-to-leucine mutation (V67L) can globally enhance splicing and related cleavage reactions in minimized Mycobacterium tuberculosis RecA inteins. However, V67L mutation causes little change in crystal structures. To test whether protein dynamics contribute to activity enhancement in the V67L mutation, we have studied the conformations and dynamics of the minimized and engineered intein ΔΔIhh-V67CM and a single V67L mutant, ΔΔIhh-L67CM, by solution NMR. Chemical shift perturbations established that the V67L mutation causes global changes, including changes at the N-terminus and C-terminus of the intein, which are active sites for protein splicing. The single V67L mutation significantly slows hydrogen-exchange rates globally, indicating a shift to more stable conformations and reduction in ensemble distribution. Whereas the V67L mutation causes little change for motions on the picosecond-to-nanosecond timescale, motions on the microsecond-to-millisecond timescale affect a region involving the conserved F-block histidine and C-terminal asparagine, which are residues important for C-terminal cleavage. The V67L mutation is proposed to activate splicing by reducing the ensemble distribution of the intein structure and by modifying the active sites.     

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.     

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.     

Intein-mediated ligation and cyclization of expressed proteins

Protein splicing is a posttranslational processing event that releases an internal protein sequence from a protein precursor. During the splicing process the internal protein sequence, termed an intein, embedded in the protein precursor self-catalyzes its excision and the ligation of the flanking protein regions, termed exteins. The dissection of the splicing pathway, which involves the precise cleavage and formation of peptide bonds, and the identification of key catalytic residues at the splice junctions have led to the modulation of the protein splicing process as a protein engineering tool. Novel strategies have been developed to use intein-catalyzed reactions for the production and manipulation of proteins and peptides. These new approaches have broken down the size limitation barrier of chemical synthetic methods and are less technically demanding. The purpose of this article is to describe how to use self-splicing inteins in protein semisynthesis and backbone cyclization. The first two sections of the article provide a brief review of the distinct chemical steps that underlie protein splicing and intein enabled technology.     

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.     

Investigating the endonuclease activity of four Pyrococcus abyssi inteins

Among the 14 inteins of the Pyrococcus abyssi genome, 10 harbour the LAGLIDADG motifs of dodecapeptide endonucleases. Four of these were cloned, expressed in Escherichia coli and purified to assay their potential endonuclease activity. PabRIR1-2 and PabRIR1-3 are specific endonucleases, named PI-PabI and PI-PabII, respectively, cleaving the sequence spanning their homing site. This is consistent with their size and with the relative positions and sequences of their endonuclease motifs. However, PI-PabI is 10-fold more active than PI-PabII and a discrepancy of the DNA recognition and cleavage mechanisms was observed between the two inteins. In particular, analysis of the DNA cleavage reactions by MALDI-TOF highlighted that while the cleavage of DNA by PI-PabI consists of two steps corresponding to the cleavage of each DNA strand, PI-PabII processes the two DNA strands simultaneously. Furthermore, the two inteins interact differently with DNA. In addition, we did not detect any endonuclease activity for PabLon and PabRIR1-1. Deletions in the intein sequences and mutations in the putative endonuclease motifs probably abolish this activity. Hence, inteins from the same archaebacteria, even if contained in the same host protein, did not evolve uniformly and are presumably at different stages of the invasion cycle.     

Study of protein splicing and intein-mediated peptide bond cleavage under high-cell-density conditions

Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins.     

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.     

Branched intermediate formation is the slowest step in the protein splicing reaction of the Ala1 KlbA intein from Methanococcus jannaschii

We report the first detailed investigation of the kinetics of protein splicing by the Methanococcus jannaschii KlbA (Mja KlbA) intein. This intein has an N-terminal Ala in place of the nucleophilic Cys or Ser residue that normally initiates splicing but nevertheless splices efficiently in vivo [Southworth, M. W., Benner, J., and Perler, F. B. (2000) EMBO J.19, 5019-5026]. To date, the spontaneous nature of the cis splicing reaction has hindered its examination in vitro. For this reason, we constructed an Mja KlbA intein-mini-extein precursor using intein-mediated protein ligation and engineered a disulfide redox switch that permits initiation of the splicing reaction by the addition of a reducing agent such as dithiothreitol (DTT). A fluorescent tag at the C-terminus of the C-extein permits monitoring of the progress of the reaction. Kinetic analysis of the splicing reaction of the wild-type precursor (with no substitutions in known nucleophiles or assisting groups) at various DTT concentrations shows that formation of the branched intermediate from the precursor is reversible (forward rate constant of 1.5 × 10(-3) s(-1) and reverse rate constant of 1.7 × 10(-5) s(-1) at 42 °C), whereas the productive decay of this intermediate to form the ligated exteins is faster and occurs with a rate constant of 2.2 × 10(-3) s(-1). This finding conflicts with reports about standard inteins, for which Asn cyclization has been assigned as the rate-determining step of the splicing reaction. Despite being the slowest step of the reaction, branched intermediate formation in the Mja KlbA intein is efficient in comparison with those of other intein systems. Interestingly, it also appears that this intermediate is protected against thiolysis by DTT, in contrast to other inteins. Evidence is presented in support of a tight coupling between the N-terminal and C-terminal cleavage steps, despite the fact that the C-terminal single-cleavage reaction occurs in variant Mja KlbA inteins in the absence of N-terminal cleavage. We posit that the splicing events in the Mja KlbA system are tightly coordinated by a network of intra- and interdomain noncovalent interactions, rendering its function particularly sensitive to minor disruptions in the intein or extein environments.     

Mechanism for intein C-terminal cleavage: a proposal from quantum mechanical calculations

Inteins are autocatalytic protein cleavage and splicing elements. A cysteine to alanine mutation at the N-terminal of inteins inhibits splicing and isolates the C-terminal cleavage reaction. Experiments indicate an enhanced C-terminal cleavage reaction rate upon decreasing the solution pH for the cleavage mutant, which cannot be explained by the existing mechanistic framework. We use intein crystal structure data and the information about conserved amino acids to perform semiempirical PM3 calculations followed by high-level density functional theory calculations in both gas phase and implicit solvent environments. Based on these calculations, we propose a detailed “low pH” mechanism for intein C-terminal cleavage. Water plays an important role in the proposed reaction mechanism, acting as an acid as well as a base. The protonation of the scissile peptide bond nitrogen by a hydronium ion is an important first step in the reaction. That step is followed by the attack of the C-terminal asparagine side chain on its carbonyl carbon, causing succinimide formation and simultaneous peptide bond cleavage. The computed reaction energy barrier in the gas phase is ~33 kcal/mol and reduces to ~25 kcal/mol in solution, close to the 21 kcal/mol experimentally observed at pH 6.0. This mechanism is consistent with the observed increase in C-terminal cleavage activity at low pH for the cleavage mutant of the Mycobacterium tuberculosis RecA mini-intein.     

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.     

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.     

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.     

Evaluation and comparison of protein splicing by exogenous inteins with foreign exteins in Escherichia coli

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

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。     

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.     

Evolution,Mechanisms, and Applications of Intein-mediated Protein Splicing

Intein-mediated protein splicing raises questions and creates opportunities in many scientific areas. Evolutionary biologists question whether inteins are primordial enzymes or simply selfish elements, whereas biochemists seek to understand how inteins work. Synthetic chemists exploit inteins in the semisynthesis of proteins with or without nonribosomal modifications, whereas biotechnologists use modified inteins in an ever increasing variety of applications. The four minireviews in this series explore these themes. The first minireview focuses on the evolution and biological function of inteins, whereas the second describes the mechanisms that underlie the remarkable ability of inteins to perform complex sets of choreographed enzymatic reactions. The third explores the relationship between the three-dimensional structure and dynamics of inteins and their biochemical capabilities. The fourth describes intein applications that have moved beyond simple technology development to utilizing inteins in more sophisticated applications, such as biosensors for identifying ligands of human hormone receptors or improved methods of biofuel and plant-based sugar production.