Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94. I. Evidence for allosteric regulation of ligand binding.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Rosser, MF; Nicchitta, CV

Published Date

  • July 28, 2000

Published In

Volume / Issue

  • 275 / 30

Start / End Page

  • 22798 - 22805

PubMed ID

  • 10816561

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M001477200


  • eng

Conference Location

  • United States