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Biomedical subjects

G Ghosh

Publications and source records attributed to G Ghosh.

At least 19 recordsLinked to original sources
PubMed1998

Crystal structure of p50/p65 heterodimer of transcription factor NF-kappaB bound to DNA.

The NF-kappaB p50/p65 heterodimer is the classical member of the Rel family of transcription factors which regulate diverse cellular functions such as immune response, cell growth, and development. Other mammalian Rel family members, including the proteins p52, proto-oncoprotein c-Rel, and RelB, all have amino-terminal Rel-homology regions (RHRs). The RHR is responsible for the dimerization, DNA binding and cytosolic localization of these proteins by virtue of complex formation with inhibitor kappaB proteins. Signal-induced removal of kappaB inhibitors allows translocation of dimers to the cell nucleus and transcriptional regulation of kappaB DNA-containing genes. NF-kappaB specifically recognizes kappaB DNA elements with a consensus sequence of 5'-GGGRNYYYCC-3' (R is an unspecified purine; Y is an unspecified pyrimidine; and N is any nucleotide). Here we report the crystal structure at 2.9 A resolution of the p50/p65 heterodimer bound to the kappaB DNA of the intronic enhancer of the immunoglobulin light-chain gene. Our structure reveals a 5-base-pair 5' subsite for p50, and a 4-base-pair 3' subsite for p65. This structure indicates why the p50/p65 heterodimer interface is stronger than that of either homodimer. A comparison of this structure with those of other Rel dimers reveals that both subunits adopt variable conformations in a DNA-sequence-dependent manner. Our results explain the different behaviour of the p50/p65 heterodimer with heterologous promoters.

Amino Acid Sequence↗
PubMed1998

A novel DNA recognition mode by the NF-kappa B p65 homodimer.

The crystal structure of the NF-kappa B p65 (RelA) homodimer in complex with a DNA target has been determined to 2.4 A resolution. The two p65 subunits are not symmetrically disposed on the DNA target. The homodimer should optimally bind to a pseudo-palindromic nine base pair target with each subunit recognizing a 5'GGAA-3' half site separated by a central A-T base pair. However, one of the subunits (subunit B) encounters a half site of 5'-GAAA-3'. The single base-pair change from G-C to A-T results in highly unfavorable interactions between this half site and the base contacting protein residues in subunit B, which leads to an 18 degrees rotation of the N-terminal terminal domain from its normal conformation. Remarkably, subunit B retains all the interactions with the sugar phosphate backbone of the DNA target. This mode of interaction allows the NF-kappa B p65 homodimer to recognize DNA targets containing only one cognate half site. Differences in the sequence of the other half site provide variations in conformation and affinity of the complex.

Amino Acid Sequence↗
PubMed1997

The role of DNA in the mechanism of NFkappaB dimer formation: crystal structures of the dimerization domains of the p50 and p65 subunits.

BACKGROUND: Members of the rel/NFkappaB family of transcription factors play a vital role in the regulation of rapid cellular responses, such as those required to fight infection or react to cellular stress. Members of this family of proteins form homo- and heterodimers with differing affinities for dimerization. They share a structural motif known as the rel homology region (RHR), the C-terminal one third of which mediates protein dimerization. Crystal structures of the rel/NFkappaB family members p50 and p65 in their DNA-bound homodimeric form have been solved. These structures showed that the residues from the dimerization domains of both p50 and p65 participate in DNA binding and that the DNA-protein and protein dimerization surfaces form one continuous overlapping interface. We desired to investigate the contribution of DNA to NFkappaB dimerization and to identify the mechanism for the selective association of rel/NFkappaB family peptides into transcriptionally active dimers. RESULTS: We report here the crystal structures of the dimerization domains of murine p50 and p65 at 2.2 A and 2.0 A resolution, respectively. A comparison of these two structures suggests that conservative amino acid changes at three positions are responsible for the differences in their dimer interfaces. The presence of the target DNA does not change the dimer interface of either protein in any significant manner. CONCLUSIONS: These two structures suggest that the rel/NFkappaB family of transcription factors use only a few conservative changes in their amino acid sequences to form a host of dimers with varying affinities for dimerization. Amino acids at positions corresponding to 254, 267, and 307 of murine p50, function as primary determinants for the observed differences in dimerization affinity. The DNA-contacting charged amino acid sidechains from the dimerization domains are held in a similar conformation in both the DNA-bound and free states, therefore, no major structural rearrangement is required to bring these residues into contact with the DNA.

Amino Acid Sequence↗
PubMed1997

The 2.1-A crystal structure of an archaeal preinitiation complex: TATA-box-binding protein/transcription factor (II)B core/TATA-box.

Archaea possess a basal transcriptional apparatus that resembles that of eukaryotes. Here we report the 2.1-A crystal structure of the archaeal transcription factor complex formed by the TATA-box-binding protein (TBP), the transcription factor IIB homolog, and a DNA target, all from the hyperthermophile Pyrococcus woesei. The overall fold of these two basal transcription factors is essentially the same as that of their eukaryotic counterparts. However, in comparison with the eukaryotic complexes, the archaeal TBP-DNA interface is more symmetrical, and in this structure the orientation of the preinitiation complex assembly on the promoter is inverted with respect to that seen in all crystal structures of comparable eukaryotic systems. This study of the structural details of an archaeal transcription factor complex presents the opportunity to examine the evolution of the basal eukaryotic transcriptional apparatus from a stereochemical viewpoint and to extend our understanding of the physical biochemistry of transcriptional initiation.

Amino Acid Sequence↗
PubMed1996

Structure of the oligomerization and L-arginine binding domain of the arginine repressor of Escherichia coli.

The structure of the oligomerization and L-arginine binding domain of the Escherichia coli arginine repressor (ArgR) has been determined using X-ray diffraction methods at 2.2 A resolution with bound arginine and at 2.8 A in the unliganded form. The oligomeric core is a 3-fold rotationally symmetric hexamer formed from six identical subunits corresponding to the 77 C-terminal residues (80 to 156) of ArgR. Each subunit has an alpha/beta fold containing a four-stranded antiparallel beta-sheet and two antiparallel alpha-helices. The hexamer is formed from two trimers, each with tightly packed hydrophobic cores. In the absence of arginine, the trimers stack back-to-back through a dyad-symmetric, sparsely packed hydrophobic interface. Six molecules of arginine bind at the trimer-trimer interface, each making ten hydrogen bonds to the protein including a direct ion pair that crosslinks the two protein trimers. Solution experiments with wild-type ArgR and oligomerization domain indicate that the hexameric form is greatly stabilized upon arginine binding. The crystal structures and solution experiments together suggest possible mechanisms of how arginine activates ArgR to bind to its DNA targets and provides a stereochemical basis for interpreting the results of mutagenesis and biochemical experiments with ArgR.

Amino Acid Sequence↗
PubMed1995

Structure of NF-kappa B p50 homodimer bound to a kappa B site.

The 2.3-A crystal structure of the transcription factor NK-kappa B p50 homodimer bound to a palindromic kappa B site reveals that the Rel homology region folds into two distinct domains, similar to those in the immunoglobulin superfamily. The p50 dimer envelopes an undistorted B-DNA helix, making specific contacts along the 10-base-pair kappa B recognition site mainly through loops connecting secondary structure elements in both domains. The carboxy-terminal domains form a dimerization interface between beta-sheets using residues that are strongly conserved in the Rel family.

Amino Acid Sequence↗
PubMed1995

Parenteral ciprofloxacin in persistent diarrhoea in children.

In a controlled trial of 30 cases of persistent diarrhoea in infants, ciprofloxacin was used parenterally for systemic antibiotic therapy. Group A cases (15 in number) were treated with parenteral ciprofloxacin and group B control cases (remaining 15) were treated with parenteral ampicillin and chloramphenicol or ampicillin/amikacin along with treatment of dehydration and malnutrition. All group A cases had excellent response and 9 infants of group B had no response up to day 4. After stopping all other drugs and with parenteral ciprofloxacin they responded favourably. Follow-up of all the cases was done regularly every 2 weeks for 6 months after discharge. No toxicity of ciprofloxacin was noted.

Anti-Bacterial Agents↗
PubMed1994

An in vitro characterization of cefditoren, a new oral cephalosporin.

Cefditoren (ME 1206) is a new cephalosporin available for oral administration as the pivaloyloxymethyl ester (ME 1207). The effect of medium formulation. pH, cation concentration and inoculum on the in vitro activity of cefditoren was investigated prior to determining its comparative antibacterial potency against a wide range of clinical bacterial isolates, its bactericidal activity against susceptible strains and the duration of its post-antibiotic effect (PAE). Cefditoren was shown to possess a broad-spectrum of cidal antibacterial activity against both Gram-positive and Gram-negative species with stability to many beta-lactamases of clinical importance. Its activity against Gram-positive species was similar to augmentin and cefuroxime, but superior to that of cefaclor and cefixime, while its beta-lactamase stability was similar to that of cefixime and ceftazidine, characterizing it as a third generation cephalosporin. Investigation of the effect of laboratory variables on the in vitro activity of cefditoren indicates that it will present no special problems when tested in the clinical setting against bacterial pathogens. PAE of 0.9 h, or greater, for Staphylococcus spp, Streptococcus pneumoniae and Moraxella catarrhalis may support the use of an extended dose-interval when cefditoren is used for the treatment of respiratory tract infections.

Bacteria↗
PubMed1993

The relationship between synthetic and editing functions of the active site of an aminoacyl-tRNA synthetase.

We have analyzed, by site-directed mutagenesis, the molecular basis of the editing function and its relation to the synthetic function of Escherichia coli methionyl-tRNA synthetase. The data obtained fit a model of the active site that partitions an amino acid substrate between synthetic and editing pathways. Hydrophobic and hydrogen bonding interactions direct the cognate substrate methionine through the synthetic pathway and prevent it from entering the editing pathway. Two hydrophobic interactions are proposed: between the side chain of Trp-305 and a methyl group of methionine and between the benzene ring of Tyr-15 and the beta- and gamma-CH2 groups of the substrate. An essential hydrogen bond forms between the OH of Tyr-15 and an electron pair of the sulfur atom of methionine. Consistent with these functions, side chains of Trp-305 and Tyr-15 are localized on opposite sides of the cavity forming a putative methionine binding pocket that is observed in the three-dimensional crystallographic structure of methionyl-tRNA synthetase. Enzymes W305A, Y15A, and Y15F have diminished ability to discriminate against homocysteine in the synthetic reaction, compared to the wild-type enzyme. At the same time, mutant enzymes have lost the ability to discriminate against methionine in the editing reaction and edited Met-AMP to a similar extent as Hcy-AMP. Interactions of residues Arg-233 and Asp-52 of methionyl-tRNA synthetase with the carboxyl and amino groups, respectively, of the substrate, which are essential for the synthetic function, were also essential for the editing function of the enzyme. Deacylation of Met-tRNA to S-methylhomocysteine thiolactone catalyzed by W305A, Y15A, and Y15F mutant enzymes was only slightly impaired relative to the wild-type enzyme. However, enzymes R233Q, R233A, and D52A did not deacylate Met-tRNA. The model also explains why the noncognate homocysteine is edited by methionyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1991

Arginine-395 is required for efficient in vivo and in vitro aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase.

We have previously shown that the anticodon of methionine tRNAs contains the major recognition site required for aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase (MetRS) and have located part of the anticodon binding domain on the enzyme at a site close to Trp461 [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768; Ghosh, G., Pelka, H., & Schulman, L.H. (1990) Biochemistry 29, 2220-2225]. In order to gain information about other possible sites of contact between MetRS and its tRNA substrates, we have examined the effects of mutations at a series of positively charged residues on the surface of the C-terminal domain of the enzyme. Conversion of Arg356, Arg366, Arg380, or Arg453 to Gln had little or no effect on enzyme activity. Similarly, conversion of Lys402 or Lys439 to Asn failed to significantly alter aminoacylation activity. Conversion of Arg380 to Ala or Arg442 to Gln produced a 5-fold reduction in kcat/Km for aminoacylation of tRNAfMet, with no effect on methionine activation, indicating a possible minor role for these residues in interaction of the enzyme with the tRNA substrate. In contrast, mutation of a phylogenetically conserved residue, Arg395, to Gln increased the Km for aminoacylation of tRNAfMet about 30-fold and reduced kcat/Km by 25,000-fold. The mutant enzyme was also shown to be highly defective by its inability to complement a strain of E. coli having an altered chromosomal MetRS gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Activation of methionine by Escherichia coli methionyl-tRNA synthetase.

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1991

Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthetase.

The crystal structure of a fully biologically active monomeric form of Escherichia coli methionyl-tRNA synthetase (MetRS) complexed with ATP has recently been reported (Brunie, S., Zelwer, C., and Risler, J.-L., (1990) J. Mol. Biol. 216, 411-424), revealing details of the active site of the enzyme, including the location of amino acid residues potentially involved in substrate binding. In the present paper, the role of 3 active site residues in interaction with methionine, ATP, and tRNA(fMet) and in catalysis of methionyl-adenylate has been explored using site-directed mutagenesis. Lys142 is located near the ribose of ATP in the MetRS.ATP cocrystal. Mutation of this residue to Ala caused a 5-fold decrease in kcat/Km for ATP-PPi exchange, indicating some contribution of the lysine side chain to the specificity of the enzyme. Mutation of Tyr359 to Ala produced a 14-fold increase in the Km for ATP with only a small (2-3-fold) change in the other kinetic parameters, indicating that the major role of this residue is in formation of the initial complex with ATP and/or in stabilization of the methionyl-adenylate reaction intermediate. Mutation of the adjacent residue Tyr358 to Ala had no effect on the Km values for methionine or ATP but produced nearly a 2000-fold decrease in the rate of ATP-PPi exchange. This mutation also dramatically reduced the rate of pyrophosphorolysis of the isolated MetRS.Met-AMP complex on addition of pyrophosphate without increasing the Km for PPi. None of the mutations affected the Km for tRNAfMet in the aminoacylation reaction. The results suggest that Tyr358 may enhance the rate of methionyl-adenylate formation by binding to the alpha-phosphate of ATP in the transition state. Interaction of Tyr358 and Tyr359 with ATP during the course of the reaction requires a significant change in the conformation of this region of the active site compared to the structure found in the MetRS.ATP complex. Such a shift is consistent with an induced-fit mechanism for methionine activation. Primary sequence comparisons of methionine-specific enzymes from yeast and bacterial sources reveals that Tyr358 is conserved in all of the known MetRS sequences.

Adenosine Triphosphate↗
PubMed1990

Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase.

We have previously shown that the anticodon of methionine tRNAs contains most, if not all, of the nucleotides required for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768]. Previous cross-linking experiments have also identified a site in the synthetase that lies within 14 A of the anticodon binding domain [Leon, O., & Schulman, L. H. (1987) Biochemistry 26, 5416-5422]. In the present work, we have carried out site-directed mutagenesis of this domain, creating conservative amino acid changes at residues that contain side chains having potential hydrogen-bond donors or acceptors. Only one of these changes, converting Trp461----Phe, had a significant effect on aminoacylation. The mutant enzyme showed an approximately 60-100-fold increase in Km for methionine tRNAs, with little or no change in the Km for methionine or ATP or in the maximal velocity of the aminoacylation reaction. Conversion of the adjacent Pro460 to Leu resulted in a smaller increase in Km for tRNA(Mets), with no change in the other kinetic parameters. Examination of the interaction of the mutant enzymes with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interactions between the Phe461 mutant and different anticodons. Km values were highest for tRNA(mMet) derivatives containing the normal anticodon wobble base C. Base substitutions at this site decreased the Km for aminoacylation by the Phe461 mutant, while increasing the Km for the wild-type enzyme and for the Leu460 mutant to values greater than 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed1990

In-vitro studies with ramoplanin (MDL 62,198): a novel lipoglycopeptide antimicrobial.

Ramoplanin is a novel lipoglycopeptide antimicrobial complex, isolated from the fermentation products of a strain of Actinoplanes sp. (ATCC 33076), which comprises three closely related polypeptides, each containing chlorinated phenyl moieties and D-mannose. The in-vitro activity of ramoplanin was compared with those of vancomycin and teicoplanin. Ramoplanin was very active against Staphylococcus spp., irrespective of methicillin susceptibility, with all isolates inhibited by 1 mg/l or less. Concentrations of vancomycin and teicoplanin required to inhibit the same population of bacteria were 4 and 16 mg/l, respectively. Ramoplanin was also very active against Streptococcus spp. (alpha- and beta-haemolytic species, Str. pneumoniae and Enterococcus faecalis, Corynebacterium spp. (including Cory. jeikeium), Listeria monocytogenes, Gardnerella vaginalis, Propionibacterium acnes and Gram-positive anaerobic bacteria, with all isolates inhibited by 2 mg/l, or less. In general, the activity of ramoplanin against these species was either equal to or only slightly less than teicoplanin and equal to or somewhat greater than vancomycin. With the exception of Bacteroides melaninogenicus and Bact. bivius, ramoplanin was not active against Gram-negative bacteria.

Anti-Bacterial Agents↗