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PMID:18492664

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Citation

Cunningham, CN, Krukenberg, KA and Agard, DA (2008) Intra- and intermonomer interactions are required to synergistically facilitate ATP hydrolysis in Hsp90. J. Biol. Chem. 283:21170-8

Abstract

Nucleotide-dependent conformational changes of the constitutively dimeric molecular chaperone Hsp90 are integral to its molecular mechanism. Recent full-length crystal structures (Protein Data Bank codes 2IOQ, 2CG9, AND 2IOP) of Hsp90 homologs reveal large scale quaternary domain rearrangements upon the addition of nucleotides. Although previous work has shown the importance of C-terminal domain dimerization for efficient ATP hydrolysis, which should imply cooperativity, other studies suggest that the two ATPases function independently. Using the crystal structures as a guide, we examined the role of intra- and intermonomer interactions in stabilizing the ATPase activity of a single active site within an intact dimer. This was accomplished by creating heterodimers that allow us to differentially mutate each monomer, probing the context in which particular residues are important for ATP hydrolysis. Although the ATPase activity of each monomer can function independently, we found that the activity of one monomer could be inhibited by the mutation of hydrophobic residues on the trans N-terminal domain (opposite monomer). Furthermore, these trans interactions are synergistically mediated by a loop on the cis middle domain. This loop contains hydrophobic residues as well as a critical arginine that provides a direct linkage to the gamma-phosphate of bound ATP. Small angle x-ray scattering demonstrates that deleterious mutations block domain closure in the presence of AMPPNP (5'-adenylyl-beta,gamma-imidodiphosphate), providing a direct linkage between structural changes and functional consequences. Together, these data indicate that both the cis monomer and the trans monomer and the intradomain and interdomain interactions cooperatively stabilize the active conformation of each active site and help explain the importance of dimer formation.

Links

PubMed PMC2475720 Online version:10.1074/jbc.M800046200

Keywords

Adenosine Triphosphatases/chemistry; Adenosine Triphosphate/chemistry; Binding Sites; Crystallography, X-Ray; Dimerization; Escherichia coli/metabolism; Gene Expression Regulation; HSP90 Heat-Shock Proteins/metabolism; Hydrolysis; Models, Biological; Molecular Conformation; Mutation; Protein Conformation; Protein Structure, Tertiary; Saccharomyces cerevisiae/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

YEAST:HSC82

enables

GO:0016887: ATP hydrolysis activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:HSC82

enables

GO:0016887: ATPase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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