Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step

A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.     

Site-directed mutagenesis of the yeast V-ATPase B subunit (Vma2p)

The B subunit of the vacuolar (H+)-ATPase (V-ATPase) has previously been shown to participate in nucleotide binding and to possess significant sequence homology with the alpha subunit of the mitochondrial F-ATPase, which forms the major portion of the noncatalytic nucleotide binding sites and contributes several residues to the catalytic sites of this complex. Based upon the recent x-ray structure of the mitochondrial F1 ATPase (Abrahams, J.P., Leslie, A.G., Lutter, R., and Walker, J.E. (1994) Nature 370,621-628), site-directed mutagenesis of the yeast VMA2 gene has been carried out in a strain containing a deletion of this gene. VMA2 encodes the yeast V-ATPase B subunit (Vma2p). Mutations at two residues postulated to be contributed by Vma2p to the catalytic site (R381S and Y352S) resulted in a complete loss of ATPase activity and proton transport, with the former having a partial effect on V-ATPase assembly. Interestingly, substitution of Phe for Tyr-352 had only minor effects on activity (15-30% inhibition), suggesting the requirement for an aromatic ring at this position. Alteration of Tyr-370, which is postulated to be near the adenine binding pocket at the noncatalytic sites, to Arg, Phe, or Ser caused a 30-50% inhibition of proton transport and ATPase activity, suggesting that an aromatic ring is not essential at this position. Finally, mutagenesis of residues in the region corresponding to the P-loop of the alpha subunit (H180K, H180G, H180D, N181V) also inhibited proton transport and ATPase activity by approximately 30-50%. None of the mutations in either the putative adenine binding pocket nor the P-loop region had any effect on the ability of Vma2p to correctly fold nor on the V-ATPase to correctly assemble. The significance of these results for the structure and function of the nucleotide binding sites on the B subunit is discussed.     

Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein

A 429 aa theoretical intein is encoded in the dnaB gene (DNA helicase) of the cyanobacterium Synechocystis sp. strain PCC6803. This intein is shown to be capable of protein splicing with or without its native exteins when tested in E. coli cells. A centrally located 275 amino acid sequence (residues 107-381) of this intein can be deleted without loss of the protein splicing activity, resulting in a functional mini-intein of 154 aa in size. Efficient in vivo protein trans-splicing was observed when this mini-intein was split into a 106 aa N-terminal fragment containing intein motifs A and B, and a 48 aa C-terminal fragment containing intein motifs F and G. These results indicate that the N- and C-terminal regions of the Ssp DnaB intein, whether covalently linked with each other or not, can come together through non-covalent interaction to form a protein splicing domain that is functionally sufficient and structurally independent from the centrally located endonuclease domain of the intein.     

Dissection of the sequence specificity of the Holliday junction endonuclease CCE1

CCE1 is a Holliday (four-way DNA) junction-specific endonuclease which resolves mitochondrial DNA recombination intermediates in Saccharomycescerevisiae. The junction-resolving enzymes are a diverse class, widely distributed in nature from viruses to higher eukaryotes. In common with most other junction-resolving enzymes, the cleavage activity of CCE1 is nucleotide sequence-dependent. We have undertaken a systematic study of the sequence specificity of CCE1, using a single-turnover kinetic assay and a panel of synthetic four-way DNA junction substrates. A tetranucleotide consensus cleavage sequence 5'-ACT downward arrowA has been identified, with specificity residing mainly at the central CT dinucleotide. Equilibrium constants for CCE1 binding to four-way junctions are unaffected by sequence variations, suggesting that substrate discrimination occurs predominantly in the transition state complex. CCE1 cuts most efficiently at the junction center, but can also cleave the DNA backbone at positions one nucleotide 3' or 5' of the point of strand exchange, suggesting a significant degree of conformational flexibility in the CCE1:junction complex. Introduction of base analogues at single sites in four-way junctions has allowed investigation of the sequence specificity of CCE1 in finer detail. In particular, the N7 moiety of the guanine base-pairing with the cytosine of the consensus sequence appears to be crucial for catalysis. The functional significance of sequence specificity in junction-resolving enzymes is discussed.     

The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton

The tight junction protein ZO-1 belongs to a family of multidomain proteins known as the membrane-associated guanylate kinase homologs (MAGUKs). ZO-1 has been demonstrated to interact with the transmembrane protein occludin, a second tight junction-specific MAGUK, ZO-2, and F-actin, although the nature and functional significance of these interactions is poorly understood. To further elucidate the role of ZO-1 within the epithelial tight junction, we have introduced epitope-tagged fragments of ZO-1 into cultured MDCK cells and identified domains critical for the interaction with ZO-2, occludin, and F-actin. A combination of in vitro and in vivo binding assays indicate that both ZO-2 and occludin interact with specific domains within the N-terminal (MAGUK-like) half of ZO-1, whereas the unique proline-rich C-terminal half of ZO-1 cosediments with F-actin. Consistent with these observations, we found that a construct encoding the N-terminal half of ZO-1 is specifically associated with tight junctions, whereas the unique C-terminal half of ZO-1 is distributed over the entire lateral surface of the plasma membrane and other actin-rich structures. In addition, we have identified a 244-amino acid domain within the N-terminal half of ZO-1, which is required for the stable incorporation of ZO-1 into the junctional complex of polarized MDCK cells. These observations suggest that one functional role of ZO-1 is to organize components of the tight junction and link them to the cortical actin cytoskeleton.     

Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein

Protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein involves four highly coordinated reactions that result in precise cleavage and formation of peptide bonds. In this study, we investigated the roles of the last N-extein residue (-1 residue) and the intein penultimate residue in modulating splicing reactions. Most of the 20 amino acid substitutions at the -1 position had no effect on overall protein splicing but could lead to significant accumulation of thioester intermediates when splicing was blocked by mutation. A subset of -1 substitutions attenuated the initiation of protein splicing and enabled us to demonstrate in vitro splicing of a mesophilic intein containing all wild-type catalytic residues. Substitutions involving the intein penultimate residue allowed modulation of the branch resolution and C-terminal cleavage reaction. Our data suggest that the N-S acyl rearrangement, which initiates splicing, may also serve as the rate-limiting step. Through appropriate amino acid substitutions, we were able to modulate splicing reactions in vitro by change in pH or temperature or addition of thiol reagents. Both insertion and deletion were tolerated in the central region of the intein although splicing or structure of the intein may have been affected.     

Asymmetrical recognition and activity of the I-SceI endonuclease on its site and on intron-exon junctions

Group I intron-encoded endonucleases represent a new class of double strand cutting endonucleases whose function is to initiate the homing of introns by generating double strand breaks in site-specific sequences. We have studied the mechanism of interaction of the I-SceI endonuclease with different DNA substrates derived from its natural site in the intron-less gene or from intron-exon junctions in the gene with an intron. We show that the enzyme recognizes its asymmetrical site with high affinity binding to the sequence corresponding to the downstream exon followed by binding to the upstream exon and catalysis of phosphodiester bond hydrolysis. Asymmetrical nicking activity is observed as an intermediate of the cleavage reaction. In the intron-containing gene, the enzyme recognizes the downstream intron-exon junction without any cleavage activity. This binding raises the possibility of a specific function of homing endonucleases in either gene expression or intron homing steps subsequent to DNA cleavage.     

Fine structure of the human ceruloplasmin gene

We characterized the genomic region corresponding to the human ceruloplasmin cDNA previously reported. Using PCR-direct sequencing methods, we determined precise intron/exon boundaries and intron-exon composition of the gene in the region. The gene region spanned about 50 kb and was composed of 19 exons and 18 introns. The lengths of exons and introns range from 107 to over 267 bp and from 0.44 to 10.0 kb, respectively. The translation initiation codon and the termination codon were located in exons 1 and 19, respectively. The nucleotide sequences of the introns were also determined in the region around the intron/exon boundaries for 24-220 bp. All the sequences around the intron/exon boundaries were consistent with the 5' and 3' consensus sequences for splice junctions of transcribed genes. Putative lariat sequences were identified between -17 and -42 nucleotides from the 3' splice junction for all 18 introns.     

Peptidylglycine alpha-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that catalyzes the first step leading to the C-terminal amidation of glycine-extended peptides. The catalytic core of PHM (PHMcc), refined to residues 42-356 of the PHM protein, was expressed at high levels in CHO (DG44) (dhfr-) cells. PHMcc has 10 cysteine residues involved in 5 disulfide linkages. Endoprotease Lys-C digestion of purified PHMcc under nonreducing conditions cleaved the protein at Lys219, indicating that the protein consists of separable N- and C-terminal domains with internal disulfide linkages, that are connected by an exposed linker region. Disulfide-linked peptides generated by sequential CNBr and pepsin treatment of radiolabeled PHMcc were separated by reverse phase HPLC and identified by Edman degradation. Three disulfide linkages occur in the N-terminal domain (Cys47-Cys186, Cys81-Cys126, and Cys114-Cys131), along with three of the His residues critical to catalytic activity (His107, His108, and His172). Two disulfide linkages (Cys227-Cys334 and Cys293-Cys315) occur in the C-terminal domain, along with the remaining two essential His residues (His242, His244) and Met314, thought to be essential in binding one of the two nonequivalent copper atoms. Substitution of Tyr79 or Tyr318 with Phe increased the Km of PHM for its peptidylglycine substrate without affecting the Vmax. Replacement of Glu313 with Asp increased the Km 8-fold and decreased the kcat 7-fold, again identifying this region of the C-terminal domain as critical to catalytic activity. Taking into account information on the copper ligands in PHM, we propose a two-domain model with a copper site in each domain that allows spatial proximity between previously described copper ligands and residues identified as catalytically important.     

Predicted structural alterations in proinsulin during its interactions with prohormone convertases

The intracellular conversion of proinsulin to insulin occurs via cleavage at the two dibasic sites: Arg31-Arg32, B chain-C-peptide (BC) junction; and Lys64-Arg65, A chain-C-peptide (CA) junction, catalyzed by the subtilisin-like prohormone convertases SPC3 (PC1/PC3) and SPC2 (PC2), respectively. In this report we propose a possible conformational variant of proinsulin that would facilitate the formation of enzyme-substrate complexes at the BC and AC junctions of proinsulin with the substrate binding groove of the two closely related convertases. Productive convertase interaction requires extended peptide conformations in both the CA junction (residues 62-67, LQKRGI) and the BC junction (residues 29-34, KTRREA) and leads to significant perturbations in the normally alpha-helical N-terminal region of the A chain and the extended C-terminal region of the B chain of the insulin moiety of proinsulin. In this model of the reactive conformation of human proinsulin, both processing sites assume positions that are relatively far apart. The C-peptide was then modeled in an unobtrusive conformation relative to the convertases and the remainder of the substrate, forming an extended loop of length approximately 40 A with a short alpha-helical segment rather than a random coil. A model of the stereochemical transformations that occur during the processing of proinsulin by SPC2 is presented.     

The in vitro ligation of bacterially expressed proteins using an intein from Methanobacterium thermoautotrophicum

The smallest known intein, found in the ribonucleoside diphosphate reductase gene of Methanobacterium thermoautotrophicum (Mth RIR1 intein), was found to splice poorly in Escherichia coli with the naturally occurring proline residue adjacent to the N-terminal cysteine of the intein. Splicing proficiency increased when this proline was replaced with an alanine residue. However, constructs that displayed efficient N- and C-terminal cleavage were created by replacing either the C-terminal asparagine or N-terminal cysteine of the intein, respectively, with an alanine. Furthermore, these constructs were used to specifically generate complementary reactive groups on protein sequences for use in ligation reactions. Reaction between an intein-generated C-terminal thioester on E. coli maltose-binding protein (43 kDa) and an intein-generated cysteine at the N terminus of either T4 DNA ligase (56 kDa) or thioredoxin (12 kDa) resulted in the ligation of the proteins through a native peptide bond. Thus the smallest of the known inteins is capable of splicing and its unique properties extend the utility of intein-mediated protein ligation to include the in vitro fusion of large, bacterially expressed proteins.     

Importance of residues carboxyl terminal relative to the cleavage site in substrates of mitochondrial processing peptidase for their specific recognition and cleavage

We previously identified distal and proximal arginine residues in the N-terminal portion and an aromatic amino acid at position 1 (P1' site3) relative to the cleavage site as important recognition signals in substrates of mitochondrial processing peptidase [Niidome, T., Kitada, S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 269, 24714-24722; Ogishima, T., Niidome, T., Shimokata, K., Kitada, S., and Ito, A. (1995) ibid. 270, 30322-30326]. To further elucidate the elements required for the specific recognition and cleavage by the enzyme, we synthesized synthetic peptides that possessed only the distal and proximal arginine residues and phenylalanine at the P1' site in a poly alanine sequence, and analyzed the processing reaction toward them. They were not cleaved by the peptidase although they inhibited the peptidase activity. However, when serine was introduced into the C-terminal portions of the sequence, processing was observed. The efficiency of the resultant peptides improved as the number of serine residues was increased. A peptide with serine or histidine at P2' and threonine at P3' was processed most efficiently. These results indicate that the processing reaction catalyzed by the peptidase depends not only on the N-terminal portion but also on the C-terminal portion from the cleavage site in the substrates.     

Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity

Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity. When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hybrid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions. Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154. Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.