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W Rüger

Publications and source records attributed to W Rüger.

At least 19 recordsLinked to original sources
PubMed2001

High resolution crystal structures of T4 phage beta-glucosyltransferase: induced fit and effect of substrate and metal binding.

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 that transfers glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine bases of phage T4 DNA. We report six X-ray structures of the substrate-free and the UDP-bound enzyme. Four also contain metal ions which activate the enzyme, including Mg(2+) in forms 1 and 2 and Mn(2+) or Ca(2+). The substrate-free BGT structure differs by a domain movement from one previously determined in another space group. Further domain movements are seen in the complex with UDP and the four UDP-metal complexes. Mg(2+), Mn(2+) and Ca(2+) bind near the beta-phosphate of the nucleotide, but they occupy slightly different positions and have different ligands depending on the metal and the crystal form. Whilst the metal site observed in these complexes with the product UDP is not compatible with a role in activating glucose transfer, it approximates the position of the positive charge in the oxocarbonium ion thought to form on the glucose moiety of the substrate during catalysis.

Allosteric Site↗
PubMed1999

T4 phage beta-glucosyltransferase: substrate binding and proposed catalytic mechanism.

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 which catalyses the transfer of glucose (Glc) from uridine diphosphoglucose (UDP-Glc) to 5-hydroxymethylcytosine (5-HMC) in double-stranded DNA. The glucosylation of T4 phage DNA is part of a phage DNA protection system aimed at host nucleases. We previously reported the first three-dimensional structure of BGT determined from crystals grown in ammonium sulphate containing UDP-Glc. In this previous structure, we did not observe electron density for the Glc moiety of UDP-Glc nor for two large surface loop regions (residues 68-76 and 109-122). Here we report two further BGT co-crystal structures, in the presence of UDP product (form I) and donor substrate UDP-Glc (form II), respectively. Form I crystals are grown in ammonium sulphate and the structure has been determined to 1.88 A resolution (R -factor 19.1 %). Form II crystals are grown in polyethyleneglycol 4000 and the structure has been solved to 2.3 A resolution (R -factor 19.8 %). The form I structure is isomorphous to our previous BGT UDP-Glc structure. The form II structure, however, has allowed us to model the two missing surface loop regions and thus provides the first complete structural description of BGT. In this low-salt crystal form, we see no electron density for the Glc moiety from UDP-Glc similar to previous observations. Biochemical data however, shows that BGT can cleave UDP-Glc in the absence of DNA acceptor, which probably accounts for the absence of Glc in our UDP-Glc substrate structures. The complete BGT structure now provides a basis for detailed modelling of a BGT HMC-DNA ternary complex. By using the structural similarity between the catalytic core of glycogen phosphorylase (GP) and BGT, we have modelled the position of the Glc moiety in UDP-Glc. From these two models, we propose a catalytic mechanism for BGT and identify residues involved in both DNA binding and in stabilizing a "flipped-out" 5-HMC nucleotide.

Bacteriophage T4↗
PubMed1999

Isolation and characterization of a cDNA clone encoding a thiol proteinase of Sarcocystis muris cyst mero' zoites (Apicomplexa).

A cDNA library of Sarcocystis muris cyst merozoites was screened using a digoxigenin-labeled probe. This probe was derived from a 504-bp polymerase-chain-reaction fragment representing part of a thiol proteinase. Several cDNA clones were isolated, one of which (PH08) consists of a nucleotide sequence of 1694 bp and encodes the complete prepropolypeptide of a cathepsin L-like proteinase. PH08 contains an open reading frame of 394 amino acid (aa) residues with a 46-residue signal sequence, which is followed by a 129-residue propeptide and 219 aa residues of the mature enzyme. Two potential glycosylation sites and a putative polyadenylation signal were also identified. The occurrence of the highly conserved interspersed ERFNIN aa motif, not found in cathepsin B-like proteinases, suggests the classification of the enzyme as a cathepsin L-like proteinase. Results worked out in this study will enable production of the recombinant thiol proteinase of S. muris cyst merozoites necessary for study of the substrate specificity as well as other biochemical parameters of this enzyme.

Amino Acid Sequence↗
PubMed1999

Characterization of a genomic region encoding the 32-kDa dense granule antigen of Sarcocystis muris (Apicomplexa).

Two subgenomic libraries constructed from Sarcocystis muris total DNA were screened with a cDNA probe, specific for a 32-kDa protein associated with the dense granules. Two clones reacted positively and were isolated, gDG 32/1 and gDG 32/2. Genomic clone gDG32/1 and part of clone gDG 32/2 have been sequenced. The composite nucleotide sequence of these genomic clones comprises 4.34 kb. It contains a 5' region of 2.14 kb, a first coding region (222 bp, exon I), a noncoding region (608 bp, intron), a second coding region (660 bp, exon II), and a 3' region of 693 bp. The upstream region shows a eukaryotic promoter structure and a consensus sequence for the 5' and 3' splicing sites. Thus the open reading frame (ORF DG32) coding for the 32-kDa protein of the dense granules of S. muris cyst merozoites is interrupted by an intron. To our knowledge, dg32 is the first sarcosporidian mosaic gene to be characterized.

Amino Acid Sequence↗
PubMed1999

Fold recognition study of alpha3-galactosyltransferase and molecular modeling of the nucleotide sugar-binding domain.

The structure and fold of the enzyme responsible for the biosynthesis of the xenotransplantation antigen, namely pig alpha3 galactosyltransferase, has been studied by means of computational methods. Secondary structure predictions indicated that alpha3-galactosyltransferase and related protein family members, including blood group A and B transferases and Forssman synthase, are likely to consist of alternating alpha-helices and beta-strands. Fold recognition studies predicted that alpha3-galactosyltransferase shares the same fold as the T4 phage DNA-modifying enzyme beta-glucosyltransferase. This latter enzyme displays a strong structural resemblance with the core of glycogen phosphorylase b. By using the three-dimensional structure of beta-glucosyltransferase and of several glycogen phosphorylases, the nucleotide binding domain of pig alpha3-galactosyltransferase was built by knowledge-based methods. Both the UDP-galactose ligand and a divalent cation were included in the model during the refinement procedure. The final three-dimensional model is in agreement with our present knowledge of the biochemistry and mechanism of alpha3-galactosyltransferases.

Amino Acid Sequence↗
PubMed1999

Overexpression, purification, and partial characterization of ADP-ribosyltransferases modA and modB of bacteriophage T4.

There is increasing experimental evidence that ADP-ribosylation of host proteins is an important means to regulate gene expression of bacteriophage T4. Surprisingly, this phage codes for three different ADP-ribosyltransferases, gene products Alt, ModA, and ModB, modifying partially overlapping sets of host proteins. While gene product Alt already has been isolated as a recombinant protein and its action on host RNA polymerases and transcription regulation have been studied, the nucleotide sequences of the two mod genes was published only recently. Their mode of action in the course of the infection cycle and the consequences of the ADP-ribosylations catalyzed by these enzymes remain to be investigated. Here we describe the cloning of the genes, the overexpression, purification, and partial characterization of ADP-ribosyltransferases ModA and ModB. Both proteins seem to act independently, and the ADP-ribosyl moieties are transferred to different sets of host proteins. While gene product ModA, similarly to the Alt protein, acts also on the alpha-subunit of host RNA polymerase, the ModB activity serves another set of proteins, one of which was identified as the S1 protein associated with the 30S subunit of the E. coli ribosomes.

Amino Acid Sequence↗
PubMed1998

Amplification of genomic DNA fragments of Sarcocystis muris (Apicomplexa) cyst merozoites encoding a thiol (cysteine) proteinase.

Parasites of the phylum Apicomplexa (Sporozoa) cause diseases such as malaria, toxoplasmosis, or intestinal coccidiosis. Invasive stages possess typical apical organelles such as dense granules that harbor a broad range of polypeptides that are believed to take part in the parasite-host cell interaction. In previous studies a 26-kDa polypeptide of dense granules from Sarcocystis muris cyst merozoites (bradyzoites) was characterized as a thiol (cysteine) proteinase. In this paper a method is demonstrated to amplify DNA fragments from genomic DNA of S. muris cyst merozoites by polymerase chain reaction, which probably code for the 26-kDa antigen.

Amino Acid Sequence↗
PubMed1998

Overexpression and structural characterization of the phage T4 protein DsbA.

The double strand binding protein A (DsbA) of bacteriophage T4 is one of several viral gene products participating in transcriptional regulation. These proteins interact or associate with the host RNA polymerase core enzyme, enabling the enzyme to successively initiate transcription at different classes of viral promoters: early, middle and late. This leads to a temporally controlled expression of the T4 gene products. The DsbA binding site overlaps the late promoter region, and DsbA binding seems to intensify transcription of late genes in vitro, possibly acting as an enhancer protein (Molecular Biology of Phage T4, Karam, 1994). To further investigate the function and structure of DsbA, we overexpressed the protein in E. coli and purified it to homogeneity. Physiological functionality of the recombinant protein was shown by gel retardation experiments and by circular dichroism (CD) spectroscopy. DsbA shows strong bands in the near UV-CD spectra. The far UV-CD spectroscopy analysis shows alpha-helices to be the main secondary structure elements. This is in agreement with secondary structure predictions. A possible helix-turn-helix motif in the center of the protein could be identified. Results from crosslinking and sedimentation analyses show that DsbA forms a dimer in solution. The thermal unfolding curve fits a dimer-two-state-folding-model, and the unfolding temperature was concentration dependent. Therefore, dimerization should supply the main portion of the free energy of stabilization of deltaG0 = 42 kJ/mol.

Binding Sites↗
PubMed1997

ADP-ribosylation and early transcription regulation by bacteriophage T4.

Bacteriophage T4 codes at least for two ADP-ribosylating activities, the 76 kDa Alt and the 24 kDa Mod gene products. The main target for both enzymes is the host RNA polymerase. We cloned and sequenced the alt gene and overexpressed the corresponding enzyme. The recombinant protein shows ADP-ribosylating activities in vitro, as had been described earlier for the native enzyme isolated from phage heads. The native as well as the recombinant protein ADP-ribosylate the alpha-subunit of RNA polymerase, but also subunits beta, beta' and sigma 70 and perform an autoribosylation reaction. Taking advantage of the pKWIII test system, constructed to measure promoter strengths in vivo, it was found that ADP-ribosylation of RNA polymerase leads to an increase of transcription from T4 early promoters up to a factor of two. In an infected host cell this should cause an enhanced expression of T4 genes. Depending on whether RNA polymerase was ADP-ribosylated or not, it initiated transcription at T4 promoters with different sequence characteristics: unribosylated RNA polymerase recognizes the early T4 promoters by an extended -10 region, whereas the ribosylated enzyme selects for T4 early promoters with an extended T4-specific and highly conserved -35 region. These results may reflect how the virus, step by step imposes its genetic program on the host cell, and in part they give a rationale for the extension of the consensus sequence observed with these promoters. We also sequenced the genomic region of the T4 mod gene and found two open reading frames coding both for proteins of approximately 24 kDa. Up to now none of the reading frames could be cloned into E. coli in an active form, making it highly probable that the ADP-ribosylation pattern inflicted by gene product Mod on host RNA polymerase is deleterious to these bacteria. Comparisons of the amino acid sequences showed significant homologies among the two reading frames. Computer analysis reveals that both Mod sequences and also the sequence of the Alt protein exhibit a structural concordance with the catalytic domains of other prokaryotic ADP-mono-ribosyltransferases such as the Pseudomonas aeruginosa exotoxin A, the cholera labile enterotoxin, the diphteria toxin, the heat labile enterotoxin A of E. coli, and pertussis toxin. We present a detailed model for T4 transcription regulation.

Adenosine Diphosphate↗
PubMed1997

Alternative splicing in PAX2 generates a new reading frame and an extended conserved coding region at the carboxy terminus.

PAX2 is a member of the PAX multigene family encoding transcription factors active in specific tissues during embryogenesis. Several PAX/Pax genes (PAX and Pax describe homologous genes in human and mice, respectively) have been shown to possess critical morphogenetic functions as identified by the analysis of mice targeted for Pax genes and the phenotype of patients heterozygous for PAX mutations. Mutations in PAX2 have been shown to be implicated in independent cases of renal-coloboma syndrome. Here, we report the characterisation of a new PAX2 isoform, viz. PAX2d, which arises because of the use of an alternative acceptor splice site within exon 12 of the PAX2 gene; this leads to a shift in the reading frame. A conserved coding region extended over the regular stop codon may emphasize the biological significance of this isoform.

Alternative Splicing↗
PubMed1996

Characterization of bacteriophage T4 early promoters in vivo with a new promoter probe vector.

We report on the construction of promoter probe vector pKWIII, useful in cloning and analyzing strong promoters for Escherichia coli RNA polymerase. Also T4 early promoters that proved to be difficult to clone with other vectors could be tested. The promoter activities obtained with this convenient and nonradioactive system largely correspond to those determined by pulse-labeling of transcripts in the same system. Results with well-characterized control promoters are in good agreement with values given by other authors. We present relative activities of several early promoters of phage T4 and compare these to promoter activities of other phages. Sorting the T4 promoters according to strength suggests the importance of distinct sequence elements to promoter functioning. They are centered around positions -52, -42, and -15.

Bacteriophage T4↗
PubMed1996

Characterization of genomic clones encoding two microneme antigens of Sarcocystis muris (Apicomplexa).

Subgenomic libraries were constructed from Sarcocystis muris total DNA. Hybridization screening with a microneme-specific cDNA probe resulted in two clones that were sequenced. The amino acid sequences deduced showed 87% homology among each other. Three different domains were recognized within both polypeptides. Domain I includes the putative N-terminal signal sequence. Domain II represents a strongly hydrophilic region, entirely homologous in the two genes. Domain III encodes the mature polypeptides with theoretical molecular masses of 15.1 kDa each. Among 28 amino acid changes in this region, 19 replacements are conservative. The putative polypeptides carried 12 conserved cysteine residues and showed homologies with plasma kallikrein, factor XI, and an antigen of Eimeria tenella. The recombinant proteins are recognized by the monoclonal antibody 3A8 directed against the 16/17-kDa microneme antigen of S. muris cystozoites. Antiserum raised against one of the purified fusion proteins cross-reacts with its counterpart and with the native 16/17-kDa band-doublet.

Amino Acid Sequence↗
PubMed1996

In vitro biosynthesis and in vivo processing of the major microneme antigen of Sarcocystis muris cyst merozoites.

The cDNA clone pSM/1.6 encoding the 26.5-kDa precursor molecule of the 16/17-kDa microneme antigen of Sarcocystis muris cyst merozoites was expressed in a cell-free translation/translocation system to study translocation of the protein across membranes. The antigen was found to be translocated across heterologous endoplasmic reticulum membranes. Translocation was accompanied by cleavage of a signal peptide to create a 23-kDa polypeptide that was completely protected from digestion with proteinase K. Pulse-chase analysis of [35S]-methionine-labeled S. muris cyst merozoites demonstrated that the 16/17-kDa antigen derived from a 23-kDa precursor molecule and that its processing occurred at between a few minutes and 2 h after biosynthesis. This leads to the conclusion that the native microneme antigen is secreted from the parasite cell via the endoplasmic reticulum. Sorting into micronemes might occur during transition through a Golgi-like structure, involving cleavage of the hydrophilic propeptide to create the mature 16/17-kDa protein.

Animals↗
PubMed1995

Overexpression, purification, and characterization of the ADP-ribosyltransferase (gpAlt) of bacteriophage T4: ADP-ribosylation of E. coli RNA polymerase modulates T4 "early" transcription.

The bacteriophage T4 Alt gene product is a component of the phage head and enters the host cell in the process of infection together with the phage DNA. It immediately ADP-ribosylates host RNA polymerase, presumably at only one of the two alpha-subunits. Transcription from T4 "early" promoters, therefore, might be catalyzed, at least in part, by an altered RNA polymerase. The T4 alt gene was cloned into the expression vector pBluescript. E. coli cells, transformed with this recombinant vector, overexpressed the 76 kDa Alt gene product, which was purified to homogeneity. The purified enzyme not only ADP-ribosylates the alpha-subunit of RNA polymerase, but also subunits beta and beta', as well as the sigma 70-factor. The recombinant enzyme behaved like the native enzyme isolated from mature phage particles. The effect of the ribosylation reaction on the transcription activity of host RNA polymerase was investigated in vivo. It results in a modulation of T4 "early" promoter strengths, presumably, in a number of cases, leading to an overexpression of T4 "early" genes. The degree of overexpression, in some cases, should reach 50%, and seems to be well dosed for each promoter, controlling an individual transcription unit.

Amino Acid Sequence↗
PubMed1994

Crystal structure of the DNA modifying enzyme beta-glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose.

Bacteriophage T4 beta-glucosyltransferase (EC 2.4.1.27) catalyses the transfer of glucose from uridine diphosphoglucose to hydroxymethyl groups of modified cytosine bases in T4 duplex DNA forming beta-glycosidic linkages. The enzyme forms part of a phage DNA protection system. We have solved and refined the crystal structure of recombinant beta-glucosyltransferase to 2.2 A resolution in the presence and absence of the substrate, uridine diphosphoglucose. The structure comprises two domains of similar topology, each reminiscent of a nucleotide binding fold. The two domains are separated by a central cleft which generates a concave surface along one side of the molecule. The substrate-bound complex reveals only clear electron density for the uridine diphosphate portion of the substrate. The UDPG is bound in a pocket at the bottom of the cleft between the two domains and makes extensive hydrogen bonding contacts with residues of the C-terminal domain only. The domains undergo a rigid body conformational change causing the structure to adopt a more closed conformation upon ligand binding. The movement of the domains is facilitated by a hinge region between residues 166 and 172. Electrostatic surface potential calculations reveal a large positive potential along the concave surface of the structure, suggesting a possible site for duplex DNA interaction.

Bacteriophage T4↗
PubMed1994

The ADP-ribosyltransferases (gpAlt) of bacteriophages T2, T4, and T6: sequencing of the genes and comparison of their products.

The Alt gene product is a component of the T4 phage head. Upon infection of the host cell, approximately 40 copies of the Alt protein enter the cell together with the viral DNA molecule. The Alt protein then ADP-ribosylates one of the two alpha-subunits of host RNA polymerase. A restriction fragment harboring the ADP-ribosyltransferase gene of bacteriophage T4 was cloned into the plasmid vector pBluescript, the nucleotide sequence was determined, and the reading frame was identified. Two M13 clone libraries, established with DNA isolated from bacteriophages T2 and T6, then were screened for the corresponding genes. The nucleotide sequences of the three alt genes and the deduced amino acid sequences were compared. Secondary structure predictions and NAD-binding studies resulted in the location of the substrate-binding site in the NH2-terminal regions of the enzymes.

Amino Acid Sequence↗