Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones

The Hsp90 family of proteins in mammalian cells consists of Hsp90 alpha and beta, Grp94, and Trap-1 (Hsp75). Radicicol, an antifungal antibiotic that inhibits various signal transduction proteins such as v-src, ras, Raf-1, an d mos, was found to bind to Hsp90, thus making it the prototype of a second class of Hsp90 inhibitors, distinct from the chemically unrelated benzoqui none ansamycins. We have used two novel methods to immobilize radicicol, al lowing for detailed analyses of drug-protein interactions. Using these two approaches, we have studied binding of the drug to N-terminal Hsp90 point m utants expressed by in vitro translation. The results point to important dr ug contacts with amino acids inside the N-terminal ATP/ADP-binding pocket r egion and show subtle differences when compared with geldanamycin binding. Radicicol binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog tha t resides in the endoplasmic reticulum. In contrast to Hsp90, binding of ra dicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain as wel l as the adjacent negatively charged region. Radicicol also specifically bi nds to yeast Hsp90, Escherichia coil HtpG, and a newly described tumor necr osis factor receptor-interacting protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90. Thus, the radicicol-binding site appears to b e specific to and is conserved in all members of the Hsp90 family of molecu lar chaperones from bacteria to mammals, but is not present in other molecu lar chaperones with nucleotide-binding domains.