The high-resolution crystal structure of a 24-kDa gyrase B fragment fromE. coli complexed with one of the most potent coumarin inhibitors, clorobiocin

The molecular chaperone Hsp90 is required for the folding and activation of a large number of substrate proteins. These are involved in essential cellular processes ranging from signal transduction to viral replication. For the activation of its substrates, Hsp90 binds and hydrolyzes ATP, which is the key driving force for conformational conversions within the dimeric chaperone. Dimerization of Hsp90 is mediated by a C-terminal dimerization site. In addition, there is a transient ATP-induced dimerization of the two N-terminal ATP-binding domains. The resulting ring-like structure is thought to be the ATPaseactive conformation. Hsp90 is a slow ATPase with a turnover number of 1 ATP/min for the yeast protein. A key question for understanding the molecular mechanism of Hsp90 is how ATP hydrolysis is regulated and linked to conformational changes. In this study, we analyzed the activation process structurally and biochemically with a view to identify the conformational limitations of the ATPase reaction cycle. We showed that the first 24 amino acids stabilize the N-terminal domain in a rigid state. Their removal confers flexibility specifically to the region between amino acids 98 and 120. Most surprisingly, the deletion of this structure results in the complete loss of ATPase activity and in increased N-terminal dimerization. Complementation assays using heterodimeric Hsp90 show that this rigid lid acts as an intrinsic kinetic inhibitor of the Hsp90 ATPase cycle preventing N-terminal dimerization in the ground state. On the other hand, this structure acts, in concert with the 24 N-terminal amino acids of the other N-terminal domain, to form an activated ATPase and thus regulates the turnover number of Hsp90. The molecular chaperone Hsp903 is required for the activation of more than 100 key cellular proteins, like kinases and transcription factors, such as steroid hormone receptors and the tumor suppressor protein p53 (1-4). Hsp90 represents about 1% of the cellular protein in eukaryotic cells, and its expression levels further increase after exposure to cellular stressors (5). The ATPase activity of Hsp90 is required for its essential cellular functions (6, 7). The importance of ATP hydrolysis for the function of Hsp90 is highlighted by the natural inhibitors geldanamycin and radicicol, which specifically target Hsp90 (8 -10) and block the activation of substrate proteins by preventing ATP binding to Hsp90 (11,12). ATP hydrolysis by Hsp90 is a complex reaction that depends on the dimeric nature of the protein, which is preserved by a C-terminal dimerization domain (3,(13)(14)(15).Attempts to elucidate the mechanistic aspects of the ATPase reaction have resulted in the definition of a cyclic reaction pathway that leads to ATP hydrolysis. ATP binding induces conformational changes that change the surface properties of Hsp90 (16,17). Subsequent investigations showed that contact formation between the two N-terminal domains of the dimeric protein results in a key conformation during the process of ATP hydrolysis (13,1...

Section: Removal Of the N-terminal Amino Acids Of Hsp90 Reveals A Segmentioning

confidence: 99%

Intrinsic Inhibition of the Hsp90 ATPase Activity

Richter

Moser

Hagn

et al. 2006

“…Novobiocin was chosen as the agent to reduce the negative supercoil density in vivo. Novobiocin inhibits the assembly of DNA gyrase (40,41) by interfering with its ATP binding capacity. This prohibits the association of two GyrAB dimers to form the biologically active tetrameric GyrA 2 B 2 form.…”

Section: Effect Of the Orientation Of Two Inserts On D/m-prior Investmentioning

confidence: 99%

Sticky DNA Formation in Vivo Alters the Plasmid Dimer/Monomer Ratio

Vetcher¹,

Wells

2004

Our discovery that plasmids containing the Friedreich's ataxia (FRDA) expanded GAA⅐TTC sequence, which forms sticky DNA, are prone to form dimers compared with monomers in vivo is the basis of an intracellular assay in Escherichia coli for this unusual DNA conformation. Sticky DNA is a single long GAA⅐GAA⅐TTC triplex formed in plasmids harboring a pair of long GAA⅐TTC repeat tracts in the direct repeat orientation. This requirement is fulfilled by either plasmid dimers of DNAs with a single trinucleotide repeat sequence tract or by monomeric DNAs containing a pair of direct repeat GAA⅐TTC sequences. DNAs harboring a single GAA⅐TTC repeat are unable to form this type of triplex conformation. An excellent correlation was observed between the ability of a plasmid to adopt the sticky triplex conformation as assayed in vitro and its propensity to form plasmid dimers relative to monomers in vivo. The variables measured that strongly influenced these measurements are as follows: length of the GAA⅐TTC insert; the extent of periodic interruptions within the repeat sequence; the orientation of the repeat inserts; and the in vivo negative supercoil density. Nitrogen mustard cross-linking studies on a family of GAA⅐TTC-containing plasmids showed the presence of sticky DNA in vivo and, thus, serves as an important bridge between the in vitro and in vivo determinations. Biochemical genetic studies on FRDA containing DNAs grown in recA or nucleotide excision repair or ruv-deficient cells showed that the in vivo properties of sticky DNA play an important role in the monomer-dimer-sticky DNA intracellular interconversions. Thus, the sticky DNA triplex exists and functions in living cells, strengthening the likelihood of its role in the etiology of FRDA.Friedreich's ataxia (FRDA), 1 an autosomal recessive neurodegenerative disease, is the most common inherited ataxia (1). The clinical and molecular biology features of FRDA have been reviewed (1-5). The FRDA gene (X25) contains seven exons and encodes a 210-amino acid protein named frataxin (1). Ninety eight percent of FRDA patients have an expanded GAA⅐TTC repeat in the first intron of the frataxin gene, and 2% have point mutations. FRDA is a typical triplet repeat disease (6) because an expansion of the repeat is found in patients; normal alleles have 6 -34 repeats of GAA⅐TTC, but the FRDA patient alleles have 66 -1700 repeats (1-3, 6). Individuals with longer GAA⅐TTC repeats (reviewed in Ref. 1) have an earlier age of onset and more severe disease manifestations.FRDA is a loss of function disease because patients with two expanded alleles have reduced levels of frataxin. Also, a reduction in the amount of the frataxin protein, a mitochondrial protein involved in iron metabolism, is because of a diminution in the abundance of the frataxin mRNA (1). This inhibition of transcription is caused by the capacity of long GAA⅐TTC repeats to form triplexes (three-stranded DNA structures) that are known to effect this inhibition (5,(7)(8)(9)(10)(11)(12). FRDA is the only triplet repeat di...

“…We used novobiocin to inhibit the assembly of active gyrase (43,44) and thus achieve a relaxation of the DNA in vivo. This should reduce the formation of unusual DNA structures and relieve the extent of founder cell loss.…”

Section: Pkd1 Unusual Dna Conformations Are Genotoxic In E Colimentioning

confidence: 99%

PKD1 Unusual DNA Conformations Are Recognized by Nucleotide Excision Repair

Bacolla

Jaworski

Connors

et al. 2001

The 2.5-kilobase pair poly(purine⅐pyrimidine) (poly(R⅐Y)) tract present in intron 21 of the polycystic kidney disease 1 (PKD1) gene has been proposed to contribute to the high mutation frequency of the gene. To evaluate this hypothesis, we investigated the growth rates of 11 Escherichia coli strains, with mutations in the nucleotide excision repair, SOS, and topoisomerase I and/or gyrase genes, harboring plasmids containing the full-length tract, six 5-truncations of the tract, and a control plasmid (pSPL3). The full-length poly(R⅐Y) tract induced dramatic losses of cell viability during the first few hours of growth and lengthened the doubling times of the populations in strains with an inducible SOS response. The extent of cell loss was correlated with the length of the poly(R⅐Y) tract and the levels of negative supercoiling as modulated by the genotype of the strains or drugs that specifically inhibited DNA gyrase or bound to DNA directly, thereby affecting conformations at specific loci. We conclude that the unusual DNA conformations formed by the PKD1 poly(R⅐Y) tract under the influence of negative supercoiling induced the SOS response pathway, and they were recognized as lesions by the nucleotide excision repair system and were cleaved, causing delays in cell division and loss of the plasmid. These data support a role for this sequence in the mutation of the PKD1 gene by stimulating repair and/or recombination functions. Polycystic kidney disease (PKD)1 encompasses a family of closely related syndromes characterized by intraparenchymal renal cysts that are lined by a single layer of epithelial cells. The several forms of PKD include autosomal dominant polycystic kidney disease (ADPKD), which is one of the most common inherited human disorders (ϳ1 per 500 worldwide). Affected individuals typically develop large cystic kidneys, but hepatic cysts, intracranial aneurysms, and cardiac valvular abnormalities are extra-renal manifestations often associated with this disorder. Approximately half of ADPKD patients develop end stage renal disease requiring renal replacement therapy and compose ϳ5% of the chronic dialysis population in the United States (reviewed in Refs. 1-5).The genetic defect in Ͼ85% of ADPKD cases is mutations in PKD1, a gene that encodes a transcript of 14 kilobases from 46 exons spanning 50 kbp on chromosome 16p13.3 (6). The 5Ј portion of the gene (exons 1-34) is duplicated with more than 95% homology in at least three other copies on chromosome 16p13.1, from which transcripts of 20, 17, and 8.5 kilobases are released (7,8). However, it is unclear whether these PKD1-like mRNAs are translated into proteins. Polycystin-1, the product of PKD1, is thought to be involved in cell-cell and cell-matrix interactions (9 -11) and in calcium-permeable non-selective cation currents (12).Genotypic analyses of PKD1 microsatellite markers revealed that cysts originate from a single cell that, in a number of cases, underwent loss of heterozygosity. These results led to the proposal that cysts form by a "two-hit...