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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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Mutations in the nucleotide binding site of Hsp90 affect  ATP binding and hydrolysis differentially. (A) Binding of ATP to  the NH2-domains of wild-type and mutant Hsp82 in the presence  and absence of GA (see Materials and Methods). Averages of  three independent experiments are shown with error bars. (B) A  typical plot of ATP hydrolysis versus time at a concentration of  0.05 mM ATP and 10 μM Hsp82. ATPase activity was measured  with [α32P]ATP at 30°C as described in Materials and Methods.  The ATPase activity of Hsp82 (closed circles) and the E33D mutant (closed triangles) is specifically inhibited by GA (open circles  and open triangles, respectively). In contrast, the E33A (closed  squares) and D79N mutants (open squares) are devoid of GA-inhibitable ATPase activity. (C) Double reciprocal plot of the  rate of ATP hydrolysis versus ATP concentration. Km values for  wild-type Hsp82 (closed circles) and for the E33D mutant (closed  triangles) were determined as 172 μM and 520 μM, respectively.
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Figure 3: Mutations in the nucleotide binding site of Hsp90 affect ATP binding and hydrolysis differentially. (A) Binding of ATP to the NH2-domains of wild-type and mutant Hsp82 in the presence and absence of GA (see Materials and Methods). Averages of three independent experiments are shown with error bars. (B) A typical plot of ATP hydrolysis versus time at a concentration of 0.05 mM ATP and 10 μM Hsp82. ATPase activity was measured with [α32P]ATP at 30°C as described in Materials and Methods. The ATPase activity of Hsp82 (closed circles) and the E33D mutant (closed triangles) is specifically inhibited by GA (open circles and open triangles, respectively). In contrast, the E33A (closed squares) and D79N mutants (open squares) are devoid of GA-inhibitable ATPase activity. (C) Double reciprocal plot of the rate of ATP hydrolysis versus ATP concentration. Km values for wild-type Hsp82 (closed circles) and for the E33D mutant (closed triangles) were determined as 172 μM and 520 μM, respectively.

Mentions: To determine the molecular basis for the loss of in vivo function of the mutant Hsp82 proteins analyzed, we expressed the full-length proteins and the respective NH2-terminal domains in soluble form in E. coli and purified them to homogeneity. Based on the available structural information, it seemed unlikely that the residue E33 contributes significantly to ATP or GA binding. Indeed, the NH2-terminal domain of Hsp82(E33A) bound ATP and GA as efficiently as the wild-type protein. In contrast, the NH2-terminal domain of Hsp82(D79N) bound ATP much less efficiently than wild-type protein (Fig. 3 A), confirming the prediction from the structural model.


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)

Mutations in the nucleotide binding site of Hsp90 affect  ATP binding and hydrolysis differentially. (A) Binding of ATP to  the NH2-domains of wild-type and mutant Hsp82 in the presence  and absence of GA (see Materials and Methods). Averages of  three independent experiments are shown with error bars. (B) A  typical plot of ATP hydrolysis versus time at a concentration of  0.05 mM ATP and 10 μM Hsp82. ATPase activity was measured  with [α32P]ATP at 30°C as described in Materials and Methods.  The ATPase activity of Hsp82 (closed circles) and the E33D mutant (closed triangles) is specifically inhibited by GA (open circles  and open triangles, respectively). In contrast, the E33A (closed  squares) and D79N mutants (open squares) are devoid of GA-inhibitable ATPase activity. (C) Double reciprocal plot of the  rate of ATP hydrolysis versus ATP concentration. Km values for  wild-type Hsp82 (closed circles) and for the E33D mutant (closed  triangles) were determined as 172 μM and 520 μM, respectively.
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Related In: Results  -  Collection

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Figure 3: Mutations in the nucleotide binding site of Hsp90 affect ATP binding and hydrolysis differentially. (A) Binding of ATP to the NH2-domains of wild-type and mutant Hsp82 in the presence and absence of GA (see Materials and Methods). Averages of three independent experiments are shown with error bars. (B) A typical plot of ATP hydrolysis versus time at a concentration of 0.05 mM ATP and 10 μM Hsp82. ATPase activity was measured with [α32P]ATP at 30°C as described in Materials and Methods. The ATPase activity of Hsp82 (closed circles) and the E33D mutant (closed triangles) is specifically inhibited by GA (open circles and open triangles, respectively). In contrast, the E33A (closed squares) and D79N mutants (open squares) are devoid of GA-inhibitable ATPase activity. (C) Double reciprocal plot of the rate of ATP hydrolysis versus ATP concentration. Km values for wild-type Hsp82 (closed circles) and for the E33D mutant (closed triangles) were determined as 172 μM and 520 μM, respectively.
Mentions: To determine the molecular basis for the loss of in vivo function of the mutant Hsp82 proteins analyzed, we expressed the full-length proteins and the respective NH2-terminal domains in soluble form in E. coli and purified them to homogeneity. Based on the available structural information, it seemed unlikely that the residue E33 contributes significantly to ATP or GA binding. Indeed, the NH2-terminal domain of Hsp82(E33A) bound ATP and GA as efficiently as the wild-type protein. In contrast, the NH2-terminal domain of Hsp82(D79N) bound ATP much less efficiently than wild-type protein (Fig. 3 A), confirming the prediction from the structural model.

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