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PubMed2001

The N-terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts.

Toc75 is an outer envelope membrane protein of chloroplasts. It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide. The N-terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts. We prepared a 45 amino-acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein-mediated system, and purified it by reverse-phase HPLC. Its identity was confirmed by N-terminal amino-acid sequencing and matrix assisted laser desorption ionization mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine. However, it did not insert into other chloroplastic lipids, such as mono- and digalactosyl diacylglycerol, and phosphatidylcholine. Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase. The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase.

Chloroplasts↗

PubMed2004

High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase.

Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.

Active Transport, Cell Nucleus↗

PubMed2002

Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies.

In this study we have developed bioluminescence-imaging strategies to noninvasively and quantitatively image protein-protein interactions in living mice by using a cooled charge-coupled device camera and split reporter technology. We validate both complementation and intein-mediated reconstitution of split firefly luciferase proteins driven by the interaction of two strongly interacting proteins, MyoD and Id. We use transient transfection of cells and image MyoD-Id interaction after induction of gene expression in cell culture and in cells implanted into living mice. Techniques to study protein-protein interactions in living subjects will allow the study of cellular networks, including signal transduction pathways, as well as development and optimization of pharmaceuticals for modulating protein-protein interactions.

Animals↗

PubMed1997

Ribonucleotide reductase in the archaeon Pyrococcus furiosus: a critical enzyme in the evolution of DNA genomes?

Ribonucleotide reductase (RNR), the enzyme responsible for deoxyribonucleotide synthesis, has been isolated from Pyrococcus furiosus, a deeply branching hyperthermophilic, strictly anaerobic archaeon. Its gene has been cloned, sequenced, and shown to harbor two insertions encoding inteins. The purified enzyme absolutely requires adenosylcobalamin for activity, a trait that defines it as a member of class II (adenosyl-cobalamin-dependent) prokaryotic RNRs. On the other hand, the archaeal RNR has significant amino acid sequence homology with class I (aerobic non-heme iron-dependent) and class III (anaerobic iron-sulfur-dependent) RNRs present in eukaryotes and bacteria, respectively. It is proposed that this enzyme may be the closest possible relative of the original RNR, which allowed the key "RNA world" to "DNA world" transition, and that the different classes of present-day RNRs are the products of divergent evolution.

Amino Acid Sequence↗

PubMed1999

Autoproteolysis in nucleoporin biogenesis.

We have molecularly characterized a proteolytic cleavage in conserved nuclear pore complex proteins. This cleavage, previously demonstrated to be essential for the biogenesis of two nuclear pore complex proteins in mammals (Nup98 and Nup96) and yeast (Nup145-N and Nup145-C), occurs between Phe and Ser residues within a highly conserved domain in a polyprotein precursor. Here, we show that a protease is not involved in the cleavage event. By using a combination of domain mapping and site-directed mutagenesis, we demonstrate that the human nuclear pore complex protein Nup98 specifically cleaves itself between F863 and S864. A region of Nup98, amino acids 715-920, is able to cleave, whereas a smaller region, amino acids 772-920, does not cleave. In addition, we have generated a Nup98 mutant that cleaves under defined conditions in vitro. Further, the two cleaved fragments of Nup98 form a complex, providing a possible mechanism whereby specific, yet low-affinity, binding between Nup98 and Nup96 is responsible for the nuclear targeting of Nup96. Although apparently unrelated evolutionarily, Nup98 has converged on an autoproteolytic biogenesis mechanism similar to that of hedgehog proteins, the inteins, and the N-terminal nucleophile proteins.

Amino Acid Sequence↗

PubMed1998

Expressed protein ligation, a novel method for studying protein-protein interactions in transcription.

Expressed protein ligation is a novel protein semi-synthesis method that permits the in vitro ligation of a chemically synthesized C-terminal segment of a protein to a recombinant N-terminal segment fused through its C terminus to an intein protein splicing element. In principle, the practical convenience of this method, combined with the expanded opportunities in protein engineering that it provides, makes it well suited for probing the molecular basis of complex processes such as transcription. Here we describe the successful application of expressed protein ligation to the approximately 600 amino acid sigma70 subunit of Escherichia coli RNA polymerase. The resulting semi-synthetic sigma70 constructs are shown to be fully functional and have been used to map the binding region of the bacteriophage T4 anti-sigma protein, AsiA, to within amino acids 567-600 of sigma70. The success of these semi-synthesis studies sets the stage for the future generation of semi-synthetic sigma70 molecules in which unnatural amino acids and biophysical probes are site-specifically incorporated in the RNA polymerase complex.

Bacterial Proteins↗

PubMed1999

Photocross-linking of the homing endonuclease PI-SceI to its recognition sequence.

PI-SceI is an intein-encoded protein that belongs to the LAGLIDADG family of homing endonucleases. According to the crystal structure and mutational studies, this endonuclease consists of two domains, one responsible for protein splicing, the other for DNA cleavage, and both presumably for DNA binding. To define the DNA binding site of PI-SceI, photocross-linking was used to identify amino acid residues in contact with DNA. Sixty-three double-stranded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions of the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm). The best cross-linking yield (approximately 30%) was obtained with an oligodeoxynucleotide with a 5-iododeoxyuridine at position +9 in the bottom strand. The subsequent analysis showed that cross-linking had occurred with amino acid His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linking was observed, indicating the specificity of the cross-linking reaction. Chemical modification of His residues in PI-SceI by diethylpyrocarbonate leads to a substantial reduction in the binding and cleavage activity of PI-SceI. This inactivation can be suppressed by substrate binding. This result further supports the finding that at least one His residue is in close contact to the DNA. Based on these and published results, conclusions are drawn regarding the DNA binding site of PI-SceI.

Base Sequence↗

PubMed2000

Probing the structure of the PI-SceI-DNA complex by affinity cleavage and affinity photocross-linking.

The PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure. To determine the spatial relationship between bases in the PI-SceI recognition sequence and selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by photocross-linking and affinity cleavage methods. Unique solvent-accessible cysteine residues were introduced into the two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid. The results suggest that an extended beta-hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate. To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein that bind DNA. The data strongly support a model of the PI-SceI-DNA complex derived from this structure.

Base Sequence↗

PubMed2003

Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis.

Biosynthesis of the molybdenum cofactor, a chelate of molybdenum or tungsten with a novel pterin, occurs in virtually all organisms including humans. In the cofactor, the metal is complexed to the unique cis-dithiolene moiety located on the pyran ring of molybdopterin. Escherichia coli molybdopterin synthase, the protein responsible for adding the dithiolene to a desulfo precursor termed precursor Z, is a dimer of dimers containing the MoaD and MoaE proteins. The sulfur used for dithiolene formation is carried in the form of a thiocarboxylate at the MoaD C terminus. Using an intein expression system for preparation of thiocarboxylated MoaD, the mechanism of the molybdopterin synthase reaction was examined. A stoichiometry of 2 molecules of thiocarboxylated MoaD per conversion of a single precursor Z molecule to molybdopterin was observed. Examination of several synthase variants bearing mutations in the MoaE subunit identified Lys-119 as a residue essential for activity and Arg-39 and Lys-126 as other residues critical for the reaction. An intermediate of the synthase reaction was identified and characterized. This intermediate remains tightly associated with the protein and is the predominant product formed by synthase containing the K126A variant of MoaE. Mass spectral data obtained from protein-bound intermediate are consistent with a monosulfurated structure that contains a terminal phosphate group similar to that present in molybdopterin.

Chromatography↗

PubMed2006

The N terminus of GTP gamma S-activated transducin alpha-subunit interacts with the C terminus of the cGMP phosphodiesterase gamma-subunit.

Dynamic regulation of G-protein signaling in the phototransduction cascade ensures the high temporal resolution of vision. In a key step, the activated alpha-subunit of transducin (Galphat-GTP) activates the cGMP phosphodiesterase (PDE) by binding the inhibitory gamma-subunit (PDEgamma). Significant progress in understanding the interaction between Galphat and PDEgamma was achieved by solving the crystal structure of the PDEgamma C-terminal peptide bound to Galphat in the transition state for GTP hydrolysis (Slep, K. C., Kercher, M. A., He, W., Cowan, C. W., Wensel, T. G., and Sigler, P. B. (2001) Nature 409, 1071-1077). However, some of the structural elements of each molecule were absent in the crystal structure. We have probed the binding surface between the PDEgamma C terminus and activated Galphat bound to guanosine 5'-O-(3-thio)-triphosphate (GTPgammaS) using a series of full-length PDEgamma photoprobes generated by intein-mediated expressed protein ligation. For each of seven PDEgamma photoprobe species, expressed protein ligation allowed one benzoyl-L-phenylalaine substitution at selected hydrophobic C-terminal positions, and the addition of a biotin affinity tag at the extreme C terminus. We have detected photocross-linking from several PDEgamma C-terminal positions to the Galphat-GTPgammaS N terminus, particularly from PDEgamma residue 73. The overall percentage of cross-linking to the Galphat-GTPgammaSN terminus was analyzed using a far Western method for examining Galphat-GTPgammaS proteolytic digestion patterns. Furthermore, mass spectrometric analysis of cross-links to Galphat from a benzoyl-phenylalanine replacement at PDEgamma position 86 localized the region of photoinsertion to Galphat N-terminal residues Galphat-(22-26). This novel Galphat/PDEgamma interaction suggests that the transducin N terminus plays an active role in signal transduction.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2007

New and easy strategy for cloning, expression, purification, and characterization of the 5S subunit of transcarboxylase from Propionibacterium f. shermanii.

Methylmalonyl CoA-oxalacetate transcarboxylase (EC 2. 1. 3. 1) from Propionibacterium f. shermanii is a biotin dependent enzyme which transfers CO2 from methylmalonyl-CoA (MMCoA) to pyruvate via a carboxylated biotin group to form oxalacetate. It is composed of three subunits, the central cylindrical hexameric 12S subunit, the outer six dimeric 5S subunit, and the twelve 1.3S linkers. We here report the cloning, sequencing, expression, and purification of the 5S subunit. The gene was identified by matching the amino acid sequence with that of deposited in the NCBI database. For cloned 5S subunit sequence shows regions of high homology with that of pyruvate carboxylase and oxaloacetate decarboxylase. The gene encoding the 5S subunit was cloned into the pTXB1 vector. The expressed 5S subunit was purified to apparent homogeneity by a single step process by using Intein mediated protein ligation (IPL) method. The cloned 5S gene encodes a protein of 505 amino acids and of M(r) 55,700.

Amino Acid Sequence↗

PubMed2005

Protein splicing: its discovery and structural insight into novel chemical mechanisms.

Protein splicing is a posttranslational cellular process, in which an intervening protein sequence (intein) is self-catalytically excised out from a nascent protein precursor and the two flanking sequences (N- and C-exteins) are ligated to produce two mature enzymes. This unique reaction was first discovered from studies of the structure and expression of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and yet comprises two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene. Subsequent studies have demonstrated that protein splicing is not unique for the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its precise reaction mechanisms from a viewpoint of structure-directed chemistry, a series of crystal structural studies has been carried out with the use of splicing-inactive and slowly spliceable precursors of VMA1 recombinants. One precursor structure revealed that the N-terminal junction of the introduced extein polypeptide forms an intermediate containing a five-membered thiazolidine ring. The other precursor structures showed spliced products with a linkage between the N- and C-extein segments. This article summarizes biochemical and structural studies on a self-catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation.

Amino Acid Sequence↗

PubMed1998

Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3.

The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http:@www.nite.go.jp.

Chromosomes, Archaeal↗

PubMed2004

Homology modeling and mutational analysis of Ho endonuclease of yeast.

Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.

Amino Acid Sequence↗

PubMed1997

Homing endonucleases: keeping the house in order.

Homing endonucleases are rare-cutting enzymes encoded by introns and inteins. They have striking structural and functional properties that distinguish them from restriction enzymes. Nomenclature conventions analogous to those for restriction enzymes have been developed for the homing endonucleases. Recent progress in understanding the structure and function of the four families of homing enzymes is reviewed. Of particular interest are the first reported structures of homing endonucleases of the LAGLIDADG family. The exploitation of the homing enzymes in genome analysis and recombination research is also summarized. Finally, the evolution of homing endonucleases is considered, both at the structure-function level and in terms of their persistence in widely divergent biological systems.

Base Sequence↗

PubMed1998

The C-terminal carboxy group of T7 RNA polymerase ensures efficient magnesium ion-dependent catalysis.

DNA and RNA polymerases use divalent metal ions for catalysis. Crystal structures of several polymerases reveal that two acidic residues are involved in coordinating two metal ions at the catalytic centre. Bacteriophage RNA polymerases contain a highly conserved C-terminus with the carboxylate positioned near the active site. We examined whether theC-terminal carboxy group of T7 RNA polymerase is important for magnesium ion-dependent catalysis. Introduction of a methyl ester or decarboxylation of the C-terminal carboxy group was achieved with an intein-based protein expression system and an elongation rate assay was developed to test the effects of the modifications. The results show that enzymes with a modified C-terminal carboxy group exhibit a magnesium ion-dependent decrease in catalytic activity.

Bacteriophage T7↗

PubMed2001

Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility.

Homing endonucleases confer mobility to their host intervening sequence, either an intron or intein, by catalyzing a highly specific double-strand break in a cognate allele lacking the intervening sequence. These proteins are characterized by their ability to bind long DNA target sites (14-40 bp) and their tolerance of minor sequence changes in these sites. A wealth of biochemical and structural data has been generated for these enzymes over the past few years. Herein we review our current understanding of homing endonucleases, including their diversity and evolution, DNA-binding and catalytic mechanisms, and attempts to engineer them to bind novel DNA substrates.

Alternative Splicing↗

PubMed2002

High resolution crystal structure of domain I of the Saccharomyces cerevisiae homing endonuclease PI-SceI.

The homing endonuclease PI-SceI from Saccharo myces cerevisiae consists of two domains. The protein splicing domain I catalyzes the excision of the mature endonuclease (intein) from a precursor protein and the religation of the flanking amino acid sequences (exteins) to a functional protein. Furthermore, domain I is involved in binding and recognition of the specific DNA substrate. Domain II of PI-SceI, the endonuclease domain, which is structurally homologous to other homing endonucleases from the LAGLIDADG family, harbors the endonucleolytic center of PI-SceI, which in vivo initiates the homing process by introducing a double-strand cut in the approximately 35 bp recognition sequence. At 1.35 A resolution, the crystal structure of PI-SceI domain I provides a detailed view of the part of the protein that is responsible for tight and specific DNA binding. A geometry-based docking of the 75 degrees bent recognition sequence to the full-length protein implies a conformational change or hinge movement of a subdomain of domain I, the tongs part, that is predicted to reach into the major groove near base pairs +16 to +18.

Binding Sites↗