Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94 II. Ligand-mediated activation of GRP94 molecular chaperone and peptide binding activity

The N-terminal domain of eukaryotic Hsp90 proteins contains a conserved ade nosine nucleotide binding pocket that also serves as the binding site for t he Hsp90 inhibitors geldanamycin and radicicol, Although this domain is ess ential for Hsp90 function, the molecular basis for adenosine nucleotide-dep endent regulation of GRP94, the endoplasmic reticulum paralog of Hsp90, rem ains to be established. We report that bis-ANS (1,1'-is(4-anilino-5-napthal enesulfonic acid), an environment sensitive fluorophore known to interact w ith nucleotide-binding domains, binds to the adenosine nucleotide-binding d omain of GRP94 and thereby activates its molecular chaperone and peptide bi nding activities. bis-ANS was observed to elicit a tertiary conformational change in GRP94 similar to that occurring upon heat shock, which also activ ates GRP94 function. bis-ANS activation of GRP94 function was efficiently b locked by radicicol, an established inhibitory ligand for the adenosine nuc leotide binding pocket. Confirmation of the N-terminal nucleotide binding p ocket as the bis-ANS-binding site was obtained following covalent incorpora tion of bis-ANS into GRP94, trypsinolysis, and sequencing of bis-ANS-labele d limit digestion products. These data identify a ligand dependent regulati on of GRP94 function and suggest a model whereby GRP94 function is regulate d through a ligand-dependent conversion of GRP94 from an inactive to an act ive conformation.