Herpes simplex virus thymidine kinase mutations associated with resistance to acyclovir: a site-directed mutagenesis study.

Mutations in the thymidine kinase (TK) gene of herpes simplex virus (HSV) may confer resistance to acyclovir (ACV). Because of the high genetic polymorphism of this gene, discriminating between mutations related to resistance and mutations related to gene polymorphism can be difficult, especially when no sensitive strain has been previously isolated from the same patient. To assess the role of the mutations located at codons 51, 77, 83, and 175, previously detected in HSV-1 clinical isolates (F. Morfin, G. Souillet, K. Bilger, T. Ooka, M. Aymard, and D. Thouvenot, J. Infect. Dis. 182:290-293, 2000), in the acquisition of resistance to ACV, four mutants with site-directed mutations at these respective codons were constructed. The enzymatic activity of the proteins, produced using both a reticulocyte lysate system and a bacterial system, was evaluated using [(3)H]thymidine as substrate. This site-directed mutagenesis revealed that mutations at codons 51, 83, and 175 induce a loss of HSV-1 TK activity and are thus clearly involved in the acquisition of resistance to ACV. On the other hand, the mutation at codon 77 does not affect enzyme activity.

Measles virus protein interactions in yeast: new findings and caveats.

Complementary DNA clones of measles virus N, N (S228Q; L229D), Ncore (N1-400), Ntail (N401-525), P, PNT (P1-230), PCT (P231-507), L, MEL (L800-2183) and EL (L1300-2183) were fused in frame downstream of the Gal4 binding domain (BD) or activating domain (AD). All but BD-L, BD-MEL and BD-EL, were detected by western blot, with additional C- and/or N-terminal truncated products in the case of BD-N, and BD-P. BD-P and BD-PNT directly activated the reporter genes, indicating that the PNT domain displays transactivating properties. In yeast two-hybrid assays, PNT and PCT domains bind to Ncore and Ntail domains, respectively, indicating that N and P interact in a head to tail orientation via two independent binding sites. BD-N (S228Q; L229D) and AD-N displayed no or poor interaction with P proteins possibly because they may not be properly folded. L binding site on P lies within the PCT domain, and two PCT binding sites lie within the L1-799 and L800-1300 regions. Thus, N to P and P to L protein interactions in measles virus shared many features with other related Paramyxoviridae. From a human cDNA library, several candidate partners of N protein were identified which all reacted with BD-Ncore, and RNA was found to bridge the N protein with one partner.

The "catalytic" triad of isocitrate dehydrogenase kinase/phosphatase from E. coli and its relationship with that found in eukaryotic protein kinases.

The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E. coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the serine/threonine protein kinase family. However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure. In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases. In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403. Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues). Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases. Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification. Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK (-) mutant strain. In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme. However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a K(m) value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme. On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected. Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family.

Isolation, cloning, and expression of a new murine zinc finger encoding gene.

With the aim of identifying genes involved in cartilage differentiation, we have used a subtractive hybridization strategy with cDNAs from a chondrocytic cell line (MC615) and mRNAs from a mesenchymal precursor cell line (10T1/2). We have isolated a cDNA clone representing a novel mouse gene. The predicted 368-amino acid protein, designated ZF-12, contains four C(2)H(2)-type zinc finger motifs and one region homologous to the LeR domain, a finger-associated structural domain. ZF-12 mRNAs are expressed during embryonic development and in different organs in adult, including rib cartilage. These data suggest that ZF-12 might play an important role not only in cartilage differentiation, but also in basic cellular processes.

The isocitrate dehydrogenase kinase/phosphatase from Escherichia coli is highly sensitive to in-vitro oxidative conditions role of cysteine67 and cysteine108 in the formation of a disulfide-bonded homodimer.

Isocitrate dehydrogenase kinase/phosphatase (IDHK/P) is a homodimeric enzyme which controls the oxidative metabolism of Escherichia coli, and exibits a high intrinsic ATPase activity. When subjected to electrophoresis under nonreducing conditions, the purified enzyme migrates partially as a dimer. The proportion of the dimer over the monomer is greatly increased by treatment with cupric 1,10 phenanthrolinate or 5,5'-dithio-bis(2-nitrobenzoic acid), and fully reversed by dithiothreitol, indicating that covalent dimerization is produced by a disulfide bond. To identify the residue(s) involved in this intermolecular disulfide-bond, each of the eight cysteines of the enzyme was individually mutated into a serine. It was found that, under nonreducing conditions, the electrophoretic patterns of all corresponding mutants are identical to that of the wild-type, except for the Cys67-->Ser which migrates exclusively as a monomer and for the Cys108-->Ser which migrates preferentially as a dimer. Furthermore, in contrast to the wild-type enzyme and all the other mutants, the Cys67-->Ser mutant still migrates as a monomer after treatment with cupric 1,10 phenanthrolinate. This result indicates that the intermolecular disulfide bond involves only Cys67 in each IDHK/P wild-type monomer. This was further supported by mass spectrum analysis of the tryptic peptides derived from either the cupric 1,10 phenanthrolinate-treated wild-type enzyme or the native Cys108-->Ser mutant, which show that they both contain a Cys67-Cys67 disulfide bond. Moreover, both the cupric 1,10 phenanthrolinate-treated wild-type enzyme and the native Cys108-->Ser mutant contain another disulfide bond between Cys356 and Cys480. Previous results have shown that this additional Cys356-Cys480 disulfide bond is intramolecular [Oudot, C., Jault, J.-M., Jaquinod, M., Negre, D., Prost, J.-F., Cozzone, A.J. & Cortay, J.-C. (1998) Eur. J. Biochem. 258, 579-585].

Cra-dependent transcriptional activation of the icd gene of Escherichia coli.

The icd gene of Escherichia coli, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified icd P1 and a newly detected second promoter, icd P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the icd control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position -76.5 within the icd P2 promoter region and (ii) three particular mutations in the C-terminal end of the alpha subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the icd P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.

FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli.

The start site of transcription of the ppsA gene, whose expression is controlled by the regulatory protein FruR in Escherichia coli, was determined by primer extension of in vivo transcripts. The interactions of the ppsA promoter with either RNA polymerase or FruR factor were analysed by the base removal method. Our results indicate that: (i) the RNA polymerase binding site has a -10 extended module but lacks its -35 hexamer; (ii) FruR binds to a target DNA region centered around position -45.5 upstream of the ppsA gene. In addition, circular permutation analysis showed that, upon binding to its site, FruR induces a sharp bend of 120 degrees in the DNA helix, which suggests a crucial involvement of FruR-induced bending in ppsA promoter activation. Direct contacts between the upstream activating DNA and RNA polymerase were studied in an in vitro transcription assay by using reconstituted RNA polymerase mutants containing Ala substitutions in C-terminal domain of their alpha subunit. The alpha[L262A], alpha[R265A] and alpha[N268A] substitutions, which caused the most drastic reduction in the FruR-mediated activation of the ppsA promoter, had previously been shown to inhibit the upstream element-mediated activation at the rrnBP1 promoter.

Inactivation of isocitrate dehydrogenase kinase/phosphatase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine is not due to the labeling of the invariant lysine residue found in the protein kinase family.

The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/phosphatase was rapidly lost after prior incubation with the ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process. About 2 mol [3H]FSBA/mol IDHK/P were bound during the time course of the inactivation. However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol. This suggested that FSBA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond. Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme insensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue. However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480.

Three-dimensional structure of the DNA-binding domain of the fructose repressor from Escherichia coli by 1H and 15N NMR.

FruR is an Escherichia coli transcriptional regulator that belongs to the LacI DNA-binding protein family. By using 1H and 15N NMR spectroscopy, we have determined the three-dimensional solution structure of the FruR N-terminal DNA-binding domain consisting of 57 amino acid residues. A total of 809 NMR-derived distances and 54 dihedral angle constraints have been used for molecular modelling with the X-PLOR program. The resulting set of calculated structures presents an average root-mean-square deviation of 0.37 A at the main-chain level for the first 47 residues. This highly defined N-terminal part of the structure reveals a similar topology for the three alpha-helices when compared to the 3D structures of LacI and PurR counterparts. The most striking difference lies in the connection between helix II and helix III, in which three additional residues are present in FruR. This connecting segment is well structured and contains a type III turn. Apart from hydrophobic interactions of non-polar residues with the core of the domain, this connecting segment is stabilised by several hydrogen bonds and by the aromatic ring stacking between Tyr19 of helix II and Tyr28 of the turn. The region containing the putative "hinge helix" (helix IV), that has been described in PurR-DNA complex to make specific base contacts in the minor groove of DNA, is unfolded. Examination of hydrogen bonds highlights the importance of homologous residues that seem to be conserved for their ability to fulfill helix N and C-capping roles in the LacI repressor family.

DNA flexibility of the UP element is a major determinant for transcriptional activation at the Escherichia coli acetate promoter.

The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method. Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase. A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter.

Definition of a consensus DNA-binding site for the Escherichia coli pleiotropic regulatory protein, FruR.

The FruR regulator of Escherichia coli controls the initiation of transcription of several operons encoding a variety of proteins involved in carbon and energy metabolism. The sequence determinants of the FruR-binding site were analysed by using 6x His-tagged FruR and a series of double-stranded randomized oligonucleotides. FruR consensus binding sites were selected and characterized by several consecutive rounds of the polymerase chain reaction-assisted binding-site selection method (BSS) using nitrocellulose-immobilized DNA-binding protein. FruR was demonstrated to require, for binding, an 8 bp left half-site motif and a 3 bp conserved right half-site with the following sequence: 5'-GNNGAATC/GNT-3'. In this sequence, the left half-site AATC/ consensus tetranucleotide is a typical motif of the DNA-binding site of the regulators of the GalR-Lacl family. On the other hand, the high degree of degeneracy found in the right half-site of this palindrome-like structure indicated that FruR, which is a tetramer in solution, interacts asymmetrically with the two half-sites of its operator. However, potentially FruR-target sites showing a high degree of symmetry were detected in 13 genes/operons. Among these, we have focused our interest on the pfkA gene, encoding phosphofructo-kinase-1, which is negatively regulated by FruR.

Recombinant N-terminal nucleotide-binding domain from mouse P-glycoprotein. Overexpression, purification, and role of cysteine 430.

Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr1 P-glyco- protein were prepared on the basis of structure predictions. Corresponding recombinant proteins were overexpressed in Escherichia coli, and the shortest one containing amino acids 395-581 exhibited the highest solubility. Insertion of an N-terminal hexahistidine tag allowed domain purification by nickel-chelate affinity chromatography. NBD1 efficiently interacted with nucleotides. Fluorescence methods showed that ATP bound at millimolar concentrations and its 2',3'-O-(2,4,6-trinitrophenyl) derivative at micromolar concentrations, while the 2'(3')-N-methylanthraniloyl derivative had intermediate affinity. Photoaffinity labeling was achieved upon irradiation with 8-azido-ATP. The domain exhibited ATPase activity with a Km for MgATP in the millimolar range, and ATP hydrolysis was competitively inhibited by micromolar 2',3'-O-(2,4,6-trinitrophenyl)-ATP. NBD1 contained a single cysteine residue, at position 430, that was derivatized with radiolabeled N-ethylmaleimide. Cysteine modification increased 6-fold the Kd for 2'(3')-N-methylanthraniloyl-ATP and prevented 8-azido-ATP photolabeling. ATPase activity was inhibited with a 5-fold increase in the Km for MgATP. The results suggest that chemical modification of Cys-430 is involved in the N-ethylmaleimide inhibition of whole P-glycoprotein by altering substrate interaction.

Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues.

The aceK gene of Escherichia coli, which encodes the isocitrate dehydrogenase kinase/phosphatase (IDH K/P), was cloned in the pQE30 expression vector to overproduce a protein tagged with six histidine residues at its N-terminus. By using a one-step chromatographic procedure, the IDH K/P was purified to near homogeneity. The IDH K/P, which contains nine Trp residues, exhibited a characteristic intrinsic tryptophan fluorescence with a low maximal emission at 326 nm. The low value of the Stern-Volmer quenching constant in the presence of acrylamide (Ksv = 2.1 M-1) indicated that the tryptophan residues were deeply buried in the protein. Furthermore, the intrinsic tryptophan fluorescence was very sensitive to the binding of nucleotide. The quenching of protein fluorescence induced by the binding of nucleotide together with an increased intrinsic fluorescence of fluorescent nucleotide analogues, methylanthraniloyl-derivatives ADP, ATP, GDP and GTP and adenosine-5'-triphosphoro-1-(5-sulfonic-acid) naphthylamidate, were used to investigate the interaction with IDH K/P. The IDH K/P dimer was shown to contain two identical nucleotide binding sites, one on each subunit, with a Kd in the range of 1.7-2.5 microM for unmodified ADP or ATP and of 2.5-3.7 microM for fluorescently labelled nucleotides. In contrast, the affinity for GDP or GTP was 10-fold lower than for adenine nucleotides. The nucleotide binding site was located within residues 315-340 by using limited proteolysis of IDH K/P by endoproteinase Lys-C. Only one main site of cleavage was obtained: the peptide bond K346-E347 which was strongly protected in the presence of ATP.

Overproduction, purification and structural characterization of the functional N-terminal DNA-binding domain of the fru repressor from Escherichia coli K-12.

A DNA fragment encoding the DNA-binding domain (amino acids 1-60) of the Escherichia coli fru transcriptional regulator was cloned into the pGEX-KT vector and expressed in frame with the fused gene encoding glutathione S-transferase. The fusion protein was purified to homogeneity by affinity chromatography on immobilized glutathione, and then cleaved with thrombin. After separation by a cation-exchange chromatography step, the DNA-binding domain exhibited proper folding, as shown by proton NMR analysis. Furthermore, it showed specific interaction with the operator region of the ace operon, as checked by gel retardation and DNA methylation-protection experiments.

In vitro asymmetric binding of the pleiotropic regulatory protein, FruR, to the ace operator controlling glyoxylate shunt enzyme synthesis.

The fruR gene of Escherichia coli, which encodes the regulatory protein FruR, was cloned in the pT7-5 expression vector so as to overproduce a protein tagged with 6 histidine residues. By using a one-step chromatographic procedure, FruR was purified to near-homogeneity. Analysis of the protein under both denaturing and nondenaturing conditions indicated that it is a tetramer with a molecular mass of about 150 kilodaltons. The positions of interference between FruR and the operator of the acetate operon were examined. The number and nature of the nucleotides essential for FruR binding were determined by several different techniques: base methylation with dimethyl sulfate, base removal by formic acid and hydrazine, uracil interference, and hydroxyl radical footprinting. It was observed that FruR asymmetrically binds to a 16-base pair DNA sequence located 170 base pairs upstream from the transcriptional start point of the ace operon.

In vitro binding of the pleiotropic transcriptional regulatory protein, FruR, to the fru, pps, ace, pts and icd operons of Escherichia coli and Salmonella typhimurium.

Evidence has been presented suggesting that the fructose repressor, FruR, is a pleiotropic transcriptional regulatory protein controlling the expression of numerous operons concerned with carbon metabolism in Escherichia coli and Salmonella typhimurium. We have conducted in vitro DNA binding studies to ascertain the nature of the DNA sequences to which FruR binds. Employing both DNA band migration retardation and DNase I footprint analyses, FruR was found to bind to two operators within the regulatory region preceding the structural genes of the fructose operon, fruB(MH)KA. These two operators, O1 and O2, comprise nearly identical palindromes of 12 bp with a half-site of TGAAAC. The binding of FruR to these inverted repeats was found to be reversed by inclusion of micromolar concentrations of fructose-1-phosphate. The two operators are located between the single putative promoter of the fructose operon and the translational initiation site of the fruB gene. Other regulated operons were shown to bind FruR to a single site upstream of the first structural gene as follows: (1) ppsA (positive regulation); (2) icd (positive regulation); (3) aceB (positive regulation); and (4) pts (negative regulation). In all cases, low concentrations of fructose-1-phosphate displaced the protein from the DNA. The binding sites were determined, and a FruR consensus sequence was established. Computer searches revealed the presence of this sequence in numerous functionally diverse operons, implying that FruR is a global transcriptional regulatory protein in enteric bacteria.

Specific interactions between the IclR repressor of the acetate operon of Escherichia coli and its operator.

The positions of interference points between the IclR repressor of the acetate operon of Escherichia coli and its specific operator were examined. The number and nature of nucleotides essential to repressor binding were determined by scanning populations of DNA previously methylated at guanine residues by dimethyl sulfate, or depurinated by treatment with formic acid, or depyrimidated by treatment with hydrazine. A total of 46 nucleotides, distributed almost equally between the two strands of the operator region, were found to be functionally important, although to a varying extent. These are clustered in two successive domains which expand from nucleotide -54 to nucleotide -27 and can organize in a palindrome-like structure containing a large proportion of A and T residues.

Regulation of the acetate operon in Escherichia coli: purification and functional characterization of the IclR repressor.

Growth of Escherichia coli on acetate requires operation of the anaplerotic sequence known as the glyoxylate bypass. In this pathway three different enzymes are activated: malate synthase, isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase which are encoded by genes aceB, aceA and aceK, respectively. These three genes are clustered, in that order, in the same acetate (ace) operon whose expression is under the transcriptional control of the iclR gene located downstream from aceK. We have cloned the iclR gene in the pKK233-2 vector which allows optimization of both transcription and translation initiation. The IclR repressor has been overproduced, then purified to homogeneity in a one-step procedure by cation exchange chromatography after ammonium sulfate fractionation. Its specific interaction with the operator/promoter region of the ace operon has been analyzed by gel retardation and DNase I footprinting experiments. The IclR repressor has been shown to recognize a 35 bp palindromic sequence which largely overlaps the -35 recognition site of RNA polymerase. Moreover, the formation of the complex between IclR and the operator/promoter region has been found to be impaired by phosphoenol pyruvate but insensitive to acetate, acetyl-CoA, pyruvate, and oxaloacetate. These results are discussed in terms of primary regulation of the expression of the ace operon.

Overproduction and characterization of the iclR gene product of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2.

The iclR gene of Escherichia coli K-12, which encodes a regulatory protein (repressor) for the aceBAK operon, is located between that operon and metH in the 91-min region of the chromosome. The iclR gene was cloned and expressed in a coupled T7 RNA polymerase/promoter system and the gene product was identified by specific binding to a fragment containing the aceBAK operator region. The iclR gene product is a polypeptide of 274 amino acids (aa) with a calculated Mr of 29,741. Comparison of the deduced IclR aa sequence to that of Salmonella typhimurium revealed that the two IclR repressors exhibit 89% identity. A possible helix-turn-helix motif characteristic of DNA-binding proteins was found within the IclR sequence. A search in protein data banks revealed that IclR has a score of similarity of 43.7% with GylR, a transcriptional regulator of the glycerol operon of Streptomyces coelicolor.