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In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis.

Obermann WM, Sondermann H, Russo AA, Pavletich NP, Hartl FU - J. Cell Biol. (1998)

Bottom Line: Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo.Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer.Our results establish Hsp90 as an ATP-dependent chaperone.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biochemistry, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.

ABSTRACT
Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis-dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.

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Requirement of full-length Hsp90 with an intact ATP-binding site for the interaction with p23 in vivo. (A) Schematic representation of the domain structure of human Hsp90 established by limited proteolysis (Stebbins et al., 1997) and of the fragments used in  the two-hybrid assay. (B) Different fragments of Hsp90 in the pAS2-1 bait vector were cotransformed in S. cerevisiae (strain Y190) with  the cDNA encoding human p23 in the pACT2 target vector (see Materials and Methods). To monitor protein–protein interactions,  cotransformants were restreaked on SD/−Trp/−Leu/−His plates containing 25 mM 3-AT and incubated for 5 d at 30°C. Cell growth  was only observed with full-length Hsp90 protein but not for any of its subfragments or for the vector control without insert. (C) Full-length wild-type Hsp90 and the mutants E47A, E47D, and D93N were analyzed for their ability to interact with p23 by two-hybrid assay  as above. Cell growth was observed for wild-type Hsp90, the E47A and E47D mutants, but not for the D93N mutant.
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Figure 5: Requirement of full-length Hsp90 with an intact ATP-binding site for the interaction with p23 in vivo. (A) Schematic representation of the domain structure of human Hsp90 established by limited proteolysis (Stebbins et al., 1997) and of the fragments used in the two-hybrid assay. (B) Different fragments of Hsp90 in the pAS2-1 bait vector were cotransformed in S. cerevisiae (strain Y190) with the cDNA encoding human p23 in the pACT2 target vector (see Materials and Methods). To monitor protein–protein interactions, cotransformants were restreaked on SD/−Trp/−Leu/−His plates containing 25 mM 3-AT and incubated for 5 d at 30°C. Cell growth was only observed with full-length Hsp90 protein but not for any of its subfragments or for the vector control without insert. (C) Full-length wild-type Hsp90 and the mutants E47A, E47D, and D93N were analyzed for their ability to interact with p23 by two-hybrid assay as above. Cell growth was observed for wild-type Hsp90, the E47A and E47D mutants, but not for the D93N mutant.

Mentions: Recent studies implicated the small acidic protein p23 as an important cofactor of Hsp90 involved in the maturation of steroid receptor molecules (Dittmar et al., 1997). Interestingly, Toft and colleagues reported that p23 interacts with Hsp90 in an ATP-dependent manner (Sullivan et al., 1997). Initially, it was difficult to reconcile this finding with the view that Hsp90 neither binds nor hydrolyzes ATP (Jakob et al., 1996; Scheibel et al., 1997). The recent structural evidence for ATP binding by Hsp82 (Prodromou et al., 1997a) together with our demonstration that Hsp90 function is dependent on ATP binding and hydrolysis in vivo provided the basis to resolve this issue. Since point mutations mapping within the first 200 residues of Hsp90 abrogate the interaction between p23 and Hsp90 (Grenert et al., 1997), we performed a two-hybrid assay to test whether the NH2-terminal domain of Hsp90 is sufficient for p23 binding. A set of human Hsp90 constructs (Fig. 5 A) in the pAS2-1 vector served as the bait and was cotransformed with human p23 into S. cerevisiae strain Y190. Surprisingly, neither of the Hsp90 deletion fragments was sufficient to interact with p23 (Fig. 5 B), although the fragments were expressed to similar levels as the full-length Hsp90 protein (Young et al., 1998). Only the full-length Hsp90 polypeptide was able to interact with p23.


In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis.

Obermann WM, Sondermann H, Russo AA, Pavletich NP, Hartl FU - J. Cell Biol. (1998)

Requirement of full-length Hsp90 with an intact ATP-binding site for the interaction with p23 in vivo. (A) Schematic representation of the domain structure of human Hsp90 established by limited proteolysis (Stebbins et al., 1997) and of the fragments used in  the two-hybrid assay. (B) Different fragments of Hsp90 in the pAS2-1 bait vector were cotransformed in S. cerevisiae (strain Y190) with  the cDNA encoding human p23 in the pACT2 target vector (see Materials and Methods). To monitor protein–protein interactions,  cotransformants were restreaked on SD/−Trp/−Leu/−His plates containing 25 mM 3-AT and incubated for 5 d at 30°C. Cell growth  was only observed with full-length Hsp90 protein but not for any of its subfragments or for the vector control without insert. (C) Full-length wild-type Hsp90 and the mutants E47A, E47D, and D93N were analyzed for their ability to interact with p23 by two-hybrid assay  as above. Cell growth was observed for wild-type Hsp90, the E47A and E47D mutants, but not for the D93N mutant.
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Related In: Results  -  Collection

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Figure 5: Requirement of full-length Hsp90 with an intact ATP-binding site for the interaction with p23 in vivo. (A) Schematic representation of the domain structure of human Hsp90 established by limited proteolysis (Stebbins et al., 1997) and of the fragments used in the two-hybrid assay. (B) Different fragments of Hsp90 in the pAS2-1 bait vector were cotransformed in S. cerevisiae (strain Y190) with the cDNA encoding human p23 in the pACT2 target vector (see Materials and Methods). To monitor protein–protein interactions, cotransformants were restreaked on SD/−Trp/−Leu/−His plates containing 25 mM 3-AT and incubated for 5 d at 30°C. Cell growth was only observed with full-length Hsp90 protein but not for any of its subfragments or for the vector control without insert. (C) Full-length wild-type Hsp90 and the mutants E47A, E47D, and D93N were analyzed for their ability to interact with p23 by two-hybrid assay as above. Cell growth was observed for wild-type Hsp90, the E47A and E47D mutants, but not for the D93N mutant.
Mentions: Recent studies implicated the small acidic protein p23 as an important cofactor of Hsp90 involved in the maturation of steroid receptor molecules (Dittmar et al., 1997). Interestingly, Toft and colleagues reported that p23 interacts with Hsp90 in an ATP-dependent manner (Sullivan et al., 1997). Initially, it was difficult to reconcile this finding with the view that Hsp90 neither binds nor hydrolyzes ATP (Jakob et al., 1996; Scheibel et al., 1997). The recent structural evidence for ATP binding by Hsp82 (Prodromou et al., 1997a) together with our demonstration that Hsp90 function is dependent on ATP binding and hydrolysis in vivo provided the basis to resolve this issue. Since point mutations mapping within the first 200 residues of Hsp90 abrogate the interaction between p23 and Hsp90 (Grenert et al., 1997), we performed a two-hybrid assay to test whether the NH2-terminal domain of Hsp90 is sufficient for p23 binding. A set of human Hsp90 constructs (Fig. 5 A) in the pAS2-1 vector served as the bait and was cotransformed with human p23 into S. cerevisiae strain Y190. Surprisingly, neither of the Hsp90 deletion fragments was sufficient to interact with p23 (Fig. 5 B), although the fragments were expressed to similar levels as the full-length Hsp90 protein (Young et al., 1998). Only the full-length Hsp90 polypeptide was able to interact with p23.

Bottom Line: Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo.Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer.Our results establish Hsp90 as an ATP-dependent chaperone.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biochemistry, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.

ABSTRACT
Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis-dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.

Show MeSH
Related in: MedlinePlus