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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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p23 binds the ATP-bound state of purified Hsp90 in  vitro, not the ADP-bound and nucleotide-free forms. Wild-type  and mutant Hsp82 proteins (200 μg each) were bound to Ni-NTA  agarose via their His6 tags and incubated for 45 min at 30°C with  yeast p23 (100 μg) and different combinations of nucleotides and  GA as indicated (lanes 2–22). In lane 1 (control) Ni-NTA beads  without bound Hsp82 were used. Protein was eluted from the  beads with imidazole and analyzed by 15% SDS-PAGE and immunoblotting for the T7 immunotag of p23 (see Materials and  Methods).
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Figure 6: p23 binds the ATP-bound state of purified Hsp90 in vitro, not the ADP-bound and nucleotide-free forms. Wild-type and mutant Hsp82 proteins (200 μg each) were bound to Ni-NTA agarose via their His6 tags and incubated for 45 min at 30°C with yeast p23 (100 μg) and different combinations of nucleotides and GA as indicated (lanes 2–22). In lane 1 (control) Ni-NTA beads without bound Hsp82 were used. Protein was eluted from the beads with imidazole and analyzed by 15% SDS-PAGE and immunoblotting for the T7 immunotag of p23 (see Materials and Methods).

Mentions: To test whether ATP binding to Hsp90 is required for the interaction with p23, two-hybrid assays were performed with the respective mutant forms of Hsp90 (Fig. 5 C). Cells containing wild-type Hsp90, Hsp90(E47D), and Hsp90(E47A) grew in a manner dependent on the interaction between Hsp90 and p23, suggesting that ATP hydrolysis by Hsp90 is not essential for the interaction with p23 measured in vivo. In contrast, no growth was observed with Hsp90(D93N) in the two-hybrid assay, indicating that Hsp90 that is defective in nucleotide binding is no more able to associate with p23. To establish this conclusion more rigorously, in vitro binding experiments with purified Hsp82 proteins and yeast p23 (Bohen, 1998; Fang et al., 1998) were performed. Full-length wild-type and mutant Hsp82 was purified from E. coli as His6EEF-tagged proteins. Yeast p23 containing an NH2-terminally located T7 immunotag was purified upon expression in E. coli. Wild-type Hsp82 or Hsp82 proteins carrying the mutations E33A, E33D or D79N were bound to Ni-NTA resin and subsequently incubated with p23 (see Materials and Methods). Binding of p23 to the immobilized Hsp82 was analyzed by SDS-PAGE and immunoblotting (Fig. 6). Significant binding to wild-type Hsp82 and Hsp82(E33D) was only observed in the presence of the nonhydrolyzable nucleotide ATPγS and was inhibited by GA. In contrast, Hsp82(E33A) was able to form a complex with p23 in the presence of hydrolyzable ATP, indicating that p23 interacts with the ATP form of Hsp90. No complex formation was observed with the D79N mutation of Hsp82, consistent with the lack of detectable nucleotide binding by this mutant in vitro. These results allow the following conclusions: p23 recognizes the ATP-bound form of Hsp90, but not the ADP-bound form or the nucleotide-free protein. As GA inhibits complex formation in the presence of ATP, the ansamycin antibiotic mimics the ADP state of Hsp90, not the ATP state, at least as far as the interaction with p23 is concerned (see Discussion). In the presence of ATP, mutant Hsp82 carrying the catalytic site mutation E33A is arrested in the p23-bound state. In contrast, wild-type Hsp82 and Hsp82(E33D) are able to cycle between the p23-bound ATP state and the p23-free ADP and non– nucleotide-bound states. Given the ATP dependence of p23 binding it might have been expected that p23 regulates the ATPase of Hsp90. Surprisingly, however, we failed to detect any effect of p23 on the ATPase activity of purified Hsp90 (data not shown).


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)

p23 binds the ATP-bound state of purified Hsp90 in  vitro, not the ADP-bound and nucleotide-free forms. Wild-type  and mutant Hsp82 proteins (200 μg each) were bound to Ni-NTA  agarose via their His6 tags and incubated for 45 min at 30°C with  yeast p23 (100 μg) and different combinations of nucleotides and  GA as indicated (lanes 2–22). In lane 1 (control) Ni-NTA beads  without bound Hsp82 were used. Protein was eluted from the  beads with imidazole and analyzed by 15% SDS-PAGE and immunoblotting for the T7 immunotag of p23 (see Materials and  Methods).
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Figure 6: p23 binds the ATP-bound state of purified Hsp90 in vitro, not the ADP-bound and nucleotide-free forms. Wild-type and mutant Hsp82 proteins (200 μg each) were bound to Ni-NTA agarose via their His6 tags and incubated for 45 min at 30°C with yeast p23 (100 μg) and different combinations of nucleotides and GA as indicated (lanes 2–22). In lane 1 (control) Ni-NTA beads without bound Hsp82 were used. Protein was eluted from the beads with imidazole and analyzed by 15% SDS-PAGE and immunoblotting for the T7 immunotag of p23 (see Materials and Methods).
Mentions: To test whether ATP binding to Hsp90 is required for the interaction with p23, two-hybrid assays were performed with the respective mutant forms of Hsp90 (Fig. 5 C). Cells containing wild-type Hsp90, Hsp90(E47D), and Hsp90(E47A) grew in a manner dependent on the interaction between Hsp90 and p23, suggesting that ATP hydrolysis by Hsp90 is not essential for the interaction with p23 measured in vivo. In contrast, no growth was observed with Hsp90(D93N) in the two-hybrid assay, indicating that Hsp90 that is defective in nucleotide binding is no more able to associate with p23. To establish this conclusion more rigorously, in vitro binding experiments with purified Hsp82 proteins and yeast p23 (Bohen, 1998; Fang et al., 1998) were performed. Full-length wild-type and mutant Hsp82 was purified from E. coli as His6EEF-tagged proteins. Yeast p23 containing an NH2-terminally located T7 immunotag was purified upon expression in E. coli. Wild-type Hsp82 or Hsp82 proteins carrying the mutations E33A, E33D or D79N were bound to Ni-NTA resin and subsequently incubated with p23 (see Materials and Methods). Binding of p23 to the immobilized Hsp82 was analyzed by SDS-PAGE and immunoblotting (Fig. 6). Significant binding to wild-type Hsp82 and Hsp82(E33D) was only observed in the presence of the nonhydrolyzable nucleotide ATPγS and was inhibited by GA. In contrast, Hsp82(E33A) was able to form a complex with p23 in the presence of hydrolyzable ATP, indicating that p23 interacts with the ATP form of Hsp90. No complex formation was observed with the D79N mutation of Hsp82, consistent with the lack of detectable nucleotide binding by this mutant in vitro. These results allow the following conclusions: p23 recognizes the ATP-bound form of Hsp90, but not the ADP-bound form or the nucleotide-free protein. As GA inhibits complex formation in the presence of ATP, the ansamycin antibiotic mimics the ADP state of Hsp90, not the ATP state, at least as far as the interaction with p23 is concerned (see Discussion). In the presence of ATP, mutant Hsp82 carrying the catalytic site mutation E33A is arrested in the p23-bound state. In contrast, wild-type Hsp82 and Hsp82(E33D) are able to cycle between the p23-bound ATP state and the p23-free ADP and non– nucleotide-bound states. Given the ATP dependence of p23 binding it might have been expected that p23 regulates the ATPase of Hsp90. Surprisingly, however, we failed to detect any effect of p23 on the ATPase activity of purified Hsp90 (data not shown).

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