Conformations of the nucleotide and polypeptide binding domains of a cytosolic Hsp70 molecular chaperone are coupled

Katie L. Fung, Lutz Hilgenberg, Miau-Yaun Wang, William J. Chirico

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

70-kDa heat shock protein (Hsp70) molecular chaperones are ATPases that participate in protein folding by regulating protein-protein interactions. ATP binds to the highly conserved amino-terminal domain, whereas polypeptides bind to the less conserved carboxyl-terminal domain. These domains are functionally coupled. Polypeptides were previously shown to dissociate from Hsp70s upon ATP binding and to stimulate ATPase activity. We probed the structure of the yeast cytosolic Hsp70 Ssa1p using limited proteolysis to determine whether the conformations of its nucleotide and polypeptide binding domains are also coupled. Ssalp adopted three distinct conformations, nucleotide-free, ADP-dependent, and ATP-dependent. Complete conformational changes required K+ and Mg2+. Using amino-terminal sequencing, ATP- agarose chromatography, and a carboxyl-terminal-specific antibody, we mapped the locations of the major proteolytic fragments. Nucleotides altered the conformations of both the nucleotide and polypeptide binding domains. Similarly, a polypeptide altered the conformations of both domains. These results indicate that the conformations of the nucleotide and polypeptide binding domains are coupled.

Original languageEnglish
Pages (from-to)21559-21565
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number35
DOIs
Publication statusPublished - 1996 Sep 16

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Molecular Chaperones
Conformations
Nucleotides
Peptides
Adenosine Triphosphate
Adenosine Triphosphatases
Proteolysis
Protein folding
Agarose Chromatography
HSP70 Heat-Shock Proteins
Protein Folding
Chromatography
Yeast
Adenosine Diphosphate
Proteins
Yeasts
Antibodies

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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abstract = "70-kDa heat shock protein (Hsp70) molecular chaperones are ATPases that participate in protein folding by regulating protein-protein interactions. ATP binds to the highly conserved amino-terminal domain, whereas polypeptides bind to the less conserved carboxyl-terminal domain. These domains are functionally coupled. Polypeptides were previously shown to dissociate from Hsp70s upon ATP binding and to stimulate ATPase activity. We probed the structure of the yeast cytosolic Hsp70 Ssa1p using limited proteolysis to determine whether the conformations of its nucleotide and polypeptide binding domains are also coupled. Ssalp adopted three distinct conformations, nucleotide-free, ADP-dependent, and ATP-dependent. Complete conformational changes required K+ and Mg2+. Using amino-terminal sequencing, ATP- agarose chromatography, and a carboxyl-terminal-specific antibody, we mapped the locations of the major proteolytic fragments. Nucleotides altered the conformations of both the nucleotide and polypeptide binding domains. Similarly, a polypeptide altered the conformations of both domains. These results indicate that the conformations of the nucleotide and polypeptide binding domains are coupled.",
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Conformations of the nucleotide and polypeptide binding domains of a cytosolic Hsp70 molecular chaperone are coupled. / Fung, Katie L.; Hilgenberg, Lutz; Wang, Miau-Yaun; Chirico, William J.

In: Journal of Biological Chemistry, Vol. 271, No. 35, 16.09.1996, p. 21559-21565.

Research output: Contribution to journalArticle

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AU - Hilgenberg, Lutz

AU - Wang, Miau-Yaun

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AB - 70-kDa heat shock protein (Hsp70) molecular chaperones are ATPases that participate in protein folding by regulating protein-protein interactions. ATP binds to the highly conserved amino-terminal domain, whereas polypeptides bind to the less conserved carboxyl-terminal domain. These domains are functionally coupled. Polypeptides were previously shown to dissociate from Hsp70s upon ATP binding and to stimulate ATPase activity. We probed the structure of the yeast cytosolic Hsp70 Ssa1p using limited proteolysis to determine whether the conformations of its nucleotide and polypeptide binding domains are also coupled. Ssalp adopted three distinct conformations, nucleotide-free, ADP-dependent, and ATP-dependent. Complete conformational changes required K+ and Mg2+. Using amino-terminal sequencing, ATP- agarose chromatography, and a carboxyl-terminal-specific antibody, we mapped the locations of the major proteolytic fragments. Nucleotides altered the conformations of both the nucleotide and polypeptide binding domains. Similarly, a polypeptide altered the conformations of both domains. These results indicate that the conformations of the nucleotide and polypeptide binding domains are coupled.

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