The charged region of Hsp90 modulates the function of the N-terminal domain

Hsp90, an abundant heat shock protein that is highly expressed even under p hysiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors, It seems to contain two chaperone sites differing in substrate specificity . Binding of ATP or the antitumor drug geldanamycin alters the substrate af finity of the N-terminal chaperone site, whereas both substances show no in fluence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various len gths in different organisms. As this linker region represents the most stri king difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragm ent of yeast Hsp90 consisting of the N-terminal domain and the charged regi on (N272) in comparison with the isolated N-terminal domain (N210), We show that the charged region causes an increase in the affinity of the N-termin al domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affini ty for ATP and geldanamycin, whereas the ATP-binding properties of the mono meric N-terminal domain N210 are not influenced by peptide binding. We prop ose that the charged region connecting the two chaperone domains plays an i mportant role in regulating chaperone function of Hsp90.