We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-Å resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the Nand C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain N␦ atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S 3 O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.Protein splicing is a posttranslational processing event that involves the precise removal of an intervening sequence, an intein, from a protein precursor with concomitant ligation of the flanking protein sequences (N and C exteins) via a native peptide bond (1-3). In vitro splicing of inteins in heterologous proteins suggests that inteins with the first C-extein residue contain sufficient information needed for the autocatalytic splicing process without the requirement of exogenous energy or protein co-factor (4, 5).Among the more than 130 inteins identified so far, the majority contain two discrete functional domains, the homing endonuclease domain and the splicing domain (Fig. 1A) (InBase, the New England Biolabs Intein Data Base (6)). The protein splicing function of the intein does not depend on their homing endonuclease activity since splicing-proficient minimal inteins (mini-inteins) occur naturally (7-9) and have been generated by deleting the centrally located endonuclease domain. The splicing domain of a dual function intein appears to be split by the endonuclease domain into two segments, the N-and C-terminal subdomains. The N-terminal subdomain (in the range of 100 -150 residues) contains the conserved intein blocks A, B, N2, and N4, whereas the C-terminal subdomain of ϳ35-50 residues contains blocks F and G (6, 10). The N-terminal subdomain of inteins not only exhibits sequence homology with the hedgehog proteins of eucaryotes but also shares an acyl rearrangement mechanism of breaking the peptide bond preceding a nucleophile-containing residue (11). This raises the question of whether inteins share a common ancestor with other autoprocessing proteins and have evolved by acquiring a new catalytic capacity imbedde...

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A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro transsplicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the Nand C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.Protein splicing elements, termed inteins (1), catalyze their own excision from a primary translation product with the concomitant ligation of the flanking protein sequences (reviewed in Refs. 2-4). Inteins catalyze three highly coordinated reactions at the N-and C-terminal splice junctions (5, 6): 1) an acyl rearrangement at the N-terminal cysteine or serine; 2) a transesterification reaction between the two termini to form a branched ester or thioester intermediate; and 3) peptide bond cleavage coupled to cyclization of the intein C-terminal asparagine to free the intein. Inteins have been engineered to be versatile tools in protein purification (7-13), protein ligation (9, 10, 12, 14 -18), and in the formation of cyclic proteins and peptides (11,19,20). However, the ligation and cyclization approaches were limited by the need to generate an N-terminal cysteine and/or C-terminal thioester intermediate in vitro.In addition to inteins engineered to trans-splice (21-24), a naturally occurring split intein was recently identified in the dnaE gene encoding the catalytic subunit of DNA polymerase III of Synechocystis sp. PCC6803 (25). The N-terminal half of DnaE, followed by a 123-amino acid intein sequence, and the C-terminal half, preceded by a 36-amino acid intein sequence, are encoded by two open reading frames located more than 745 kilobases apart in the genome. When co-expressed in Escherichia coli, the two DnaE-intein fragments exhibited protein trans-splicing (25). In this report we have further investigated the cis-and trans-splicing activities of the Ssp DnaE intein in a foreign protein context. Furthermore, novel methods were developed that allow the on-column ligation of protein fragments as well as the in vivo and in vitro cyclization of p...

Two important aspects of protein splicing were investigated by employing the trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803. First, we demonstrated that both protein splicing and cleavage at the N-terminal splice junction were inhibited in the presence of zinc ion. The trans-splicing reaction was partially blocked at a concentration of 1-10 M Zn 2؉ and completely inhibited at 100 M Zn 2؉ ; the inhibition by zinc was reversed in the presence of ethylenediaminetetraacetic acid. We propose that inactivation of Cys 160 at the C-terminal splice junction by the chelation of zinc affects both the N-S acyl rearrangement and the transesterification steps in the splicing pathway. Furthermore, in vivo and in vitro assays were established for the determination of intein residues and regions required for splicing or association between the N-and C-terminal intein halves. N-terminal truncation of the intein C-terminal segment inhibited both splicing and association activities, suggesting this region is crucial for the formation of an interface between the two intein halves. The replacement of conserved residues in blocks B and F with alanine abolished splicing but allowed for association. This is the first evidence showing that the conserved residues in block F are required for protein splicing.Protein splicing is a post-translational processing event, which involves the self-catalyzed excision of an internal protein segment, or intein, from a protein precursor with the concomitant joining of the flanking polypeptide sequences, the exteins (1, 2). Sequence alignment reveals that an intein can be divided into three segments. The N-terminal region possesses ϳ120 -150 amino acid residues including highly conserved blocks A and B, whereas the C-terminal region is composed of ϳ35-50 residues containing conserved blocks F and G. Between the two terminal regions is an optional endonuclease domain, which has been found in a majority of inteins (3, 4). In the case of protein trans-splicing, however, a functional intein is reconstituted from two non-covalently linked N-and C-terminal segments that are separately translated (5, 6). The crystal structures of inteins from a vacuolar ATPase subunit of Saccharomyces cerevisiae (PI-SceI or Sce VMA intein), 1 Mycobacterium xenopi GyrA (Mxe GyrA intein), and Pyrococcus furiosus ribonucleotide reductase (PI-PfuI) revealed that the Nand C-terminal regions form a horseshoe-shaped Hint (hedgehog and intein) domain (7-9). The intein structure contains an unusual ␤-fold with the splice junctions at the ends of two adjacent antiparallel ␤-strands, forming a catalytic pocket. The catalytic residues implicated in the splicing mechanism have been found in the conserved blocks, A and G, present at the two splice junctions (10 -16). Presumably, these residues directly participate in protein splicing by three concerted nucleophilic replacements (1, 17) (Fig. 1). Other residues and regions that may be involved in assisting these catalytic reactions have yet to be identified, although ...

We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this procedure is that each carrier protein molecule ligates only one peptide, ensuring that the ligation product forms a sharp band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrate the effectiveness of this approach by mutational analysis of peptide substrates derived from human cyclin-dependent kinase, Cdc2, which contains a phosphorylation site of human c-Src protein tyrosine kinase.