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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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Structure of the NH2-terminal domain of Hsp90 with  bound ADP-Mg. View into the nucleotide-binding pocket. The  residues D93 and E47, critical for nucleotide binding and hydrolysis, respectively, are colored in yellow. Image was prepared with  the programs MOLSCRIPT (Kraulis, 1991) and RASTER3D  (Merrit and Murphy, 1994).
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Figure 1: Structure of the NH2-terminal domain of Hsp90 with bound ADP-Mg. View into the nucleotide-binding pocket. The residues D93 and E47, critical for nucleotide binding and hydrolysis, respectively, are colored in yellow. Image was prepared with the programs MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merrit and Murphy, 1994).

Mentions: As was observed with the structure of the NH2-terminal domain of yeast Hsp90 (Prodromou et al., 1997a), ADP-Mg binds to a site in human Hsp90 where GA also binds (Stebbins et al., 1997). The contacts between the nucleotide and Hsp90 in this complex are very similar to those observed in the yeast complex (Prodromou et al., 1997a), and involve both direct and water-mediated hydrogen bonds (Fig. 1). The N6 group of the adenine purine forms direct hydrogen bonds with the D93 side chain, and water-mediated hydrogen bonds with the S52 side chain and L48 backbone carbonyl groups. As in the case of the structure with bound GA, the interaction with D93 (D79 in yeast Hsp82) is probably most critical for stable nucleotide binding. The N7 group of the purine interacts via water-mediated hydrogen bonds with the N51 side chain, and the N1 group of the purine with the G97 backbone and D93 and T81 side chain groups. Additional ADP-Mg contacts include a hydrogen bond between the O2′ group and the N106 side chain, a set of hydrogen bonds between the α-phosphate group and the F138, G137 backbone amide and N51 side chain groups. The Mg ion is octahedrally coordinated by the α- and β-phosphate groups of the ADP, the N51 side chain of Hsp90, and three water molecules.


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)

Structure of the NH2-terminal domain of Hsp90 with  bound ADP-Mg. View into the nucleotide-binding pocket. The  residues D93 and E47, critical for nucleotide binding and hydrolysis, respectively, are colored in yellow. Image was prepared with  the programs MOLSCRIPT (Kraulis, 1991) and RASTER3D  (Merrit and Murphy, 1994).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Structure of the NH2-terminal domain of Hsp90 with bound ADP-Mg. View into the nucleotide-binding pocket. The residues D93 and E47, critical for nucleotide binding and hydrolysis, respectively, are colored in yellow. Image was prepared with the programs MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merrit and Murphy, 1994).
Mentions: As was observed with the structure of the NH2-terminal domain of yeast Hsp90 (Prodromou et al., 1997a), ADP-Mg binds to a site in human Hsp90 where GA also binds (Stebbins et al., 1997). The contacts between the nucleotide and Hsp90 in this complex are very similar to those observed in the yeast complex (Prodromou et al., 1997a), and involve both direct and water-mediated hydrogen bonds (Fig. 1). The N6 group of the adenine purine forms direct hydrogen bonds with the D93 side chain, and water-mediated hydrogen bonds with the S52 side chain and L48 backbone carbonyl groups. As in the case of the structure with bound GA, the interaction with D93 (D79 in yeast Hsp82) is probably most critical for stable nucleotide binding. The N7 group of the purine interacts via water-mediated hydrogen bonds with the N51 side chain, and the N1 group of the purine with the G97 backbone and D93 and T81 side chain groups. Additional ADP-Mg contacts include a hydrogen bond between the O2′ group and the N106 side chain, a set of hydrogen bonds between the α-phosphate group and the F138, G137 backbone amide and N51 side chain groups. The Mg ion is octahedrally coordinated by the α- and β-phosphate groups of the ADP, the N51 side chain of Hsp90, and three water molecules.

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