title: Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. I. Evidence for allosteric regulation of ligand binding.
abstract: X-ray crystallographic studies of the N-terminal domain of Hsp90 have identified an unconventional ATP binding fold, thereby inferring a role for ATP in the regulation of the Hsp90 activity. In this report, N-ethylcarboxamidoadenosine (NECA) was used to investigate the nucleotide binding properties of GRP94, the endoplasmic reticulum paralog of Hsp90. Whereas Hsp90 did not bind NECA, GRP94 bound NECA in a saturable manner with a K(d) of 200 nm. NECA binding to GRP94 was efficiently blocked by geldanamycin and radicicol. Analysis of ligand binding stoichiometries by radioligand and calorimetric techniques indicated that GRP94 bound 1 mol of NECA/mol of GRP94 dimer. In contrast, GRP94 bound radicicol at a stoichiometry of 2 mol of radicicol/mol of GRP94 dimer. In [(3)H]NECA displacement assays, GRP94 displayed binding interactions with ATP, dATP, ADP, AMP, cAMP, and adenosine, but not GTP, CTP, or UTP. To accommodate the 0.5 mol of NECA:mol of GRP94 binding stoichiometry observed for the native GRP94 dimer, a model for allosteric regulation (negative cooperativity) of ligand binding is proposed. A hypothesis on the regulation of GRP94 conformation and activity by adenosine-based ligand(s) other than ATP and ADP is presented.
authors.affiliation: Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: M F
authors.initials: MF
authors.lastname: Rosser
authors.forename: C V
authors.initials: CV
authors.lastname: Nicchitta
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats
chemical_list: D000227:Adenine Nucleotides; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D008024:Ligands; D008565:Membrane Proteins; C018551:glucose-regulated proteins; D019830:Adenosine-5'-(N-ethylcarboxamide); D000251:Adenosine Triphosphatases