Limits...
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.

Show MeSH

Related in: MedlinePlus

The complete  Hsp90 protein is required for  ATPase activity. ATP hydrolysis versus time at 0.10  mM ATP and 10 μM Hsp82  proteins at 30°C is shown for  full-length Hsp82 (FL82,  closed circles), the NH2 domain of Hsp82 (N82, closed  triangles) and for ΔC82, lacking the COOH-terminal residues 600–709 (closed squares).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132952&req=5

Figure 4: The complete Hsp90 protein is required for ATPase activity. ATP hydrolysis versus time at 0.10 mM ATP and 10 μM Hsp82 proteins at 30°C is shown for full-length Hsp82 (FL82, closed circles), the NH2 domain of Hsp82 (N82, closed triangles) and for ΔC82, lacking the COOH-terminal residues 600–709 (closed squares).

Mentions: Next, we investigated whether the stably folded NH2-terminal domain of Hsp82 (Stebbins et al., 1997; Prodromou et al., 1997a) is sufficient for the ATPase activity of Hsp82 or whether additional parts of the molecule are required for ATP hydrolysis. Surprisingly, although the NH2-terminal domain binds ATP (Fig. 3 A), it did not exhibit detectable catalytic activity in the ATPase assay (Fig. 4). Similarly, no ATPase activity was detected with a variant of Hsp82, Hsp82ΔC (Fig. 4), that lacks the COOH-terminal dimerization domain (residues 600–709; Nemoto et al., 1995; Wearsch and Nicchitta, 1996), but can be considered to be native as it was fully soluble after expression in E. coli and active in prevention of rhodanese aggregation (Young et al., 1997). Similarly, the NH2-terminal domain of Hsp82 was properly folded, as it was not only capable of binding ATP and GA (see Fig. 3 A; Prodromou et al., 1997a; Stebbins et al., 1997) but also preserved the chaperone function in preventing protein aggregation in vitro (Young et al., 1997; Scheibel et al., 1998; data not shown). Thus, the complete Hsp90 protein, presumably as the dimer, is required for ATP hydrolytic activity. Interestingly, the chaperone function of the isolated NH2-terminal domain did not exhibit a detectable dependence on specific nucleotide binding (data not shown), suggesting that ATP hydrolysis may be required for substrate release or that this function depends on the cooperation of Hsp90 with one or several of its cofactors.


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)

The complete  Hsp90 protein is required for  ATPase activity. ATP hydrolysis versus time at 0.10  mM ATP and 10 μM Hsp82  proteins at 30°C is shown for  full-length Hsp82 (FL82,  closed circles), the NH2 domain of Hsp82 (N82, closed  triangles) and for ΔC82, lacking the COOH-terminal residues 600–709 (closed squares).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132952&req=5

Figure 4: The complete Hsp90 protein is required for ATPase activity. ATP hydrolysis versus time at 0.10 mM ATP and 10 μM Hsp82 proteins at 30°C is shown for full-length Hsp82 (FL82, closed circles), the NH2 domain of Hsp82 (N82, closed triangles) and for ΔC82, lacking the COOH-terminal residues 600–709 (closed squares).
Mentions: Next, we investigated whether the stably folded NH2-terminal domain of Hsp82 (Stebbins et al., 1997; Prodromou et al., 1997a) is sufficient for the ATPase activity of Hsp82 or whether additional parts of the molecule are required for ATP hydrolysis. Surprisingly, although the NH2-terminal domain binds ATP (Fig. 3 A), it did not exhibit detectable catalytic activity in the ATPase assay (Fig. 4). Similarly, no ATPase activity was detected with a variant of Hsp82, Hsp82ΔC (Fig. 4), that lacks the COOH-terminal dimerization domain (residues 600–709; Nemoto et al., 1995; Wearsch and Nicchitta, 1996), but can be considered to be native as it was fully soluble after expression in E. coli and active in prevention of rhodanese aggregation (Young et al., 1997). Similarly, the NH2-terminal domain of Hsp82 was properly folded, as it was not only capable of binding ATP and GA (see Fig. 3 A; Prodromou et al., 1997a; Stebbins et al., 1997) but also preserved the chaperone function in preventing protein aggregation in vitro (Young et al., 1997; Scheibel et al., 1998; data not shown). Thus, the complete Hsp90 protein, presumably as the dimer, is required for ATP hydrolytic activity. Interestingly, the chaperone function of the isolated NH2-terminal domain did not exhibit a detectable dependence on specific nucleotide binding (data not shown), suggesting that ATP hydrolysis may be required for substrate release or that this function depends on the cooperation of Hsp90 with one or several of its cofactors.

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