A hydrophobic segment within the C-terminal domain is essential for both client-binding and dimer formation of the HSP90-family molecular chaperone.

The alpha isoform of human 90-kDa heat shock protein (HSP90alpha) is composed of three domains: the N-terminal (residues 1-400); middle (residues 401-615) and C-terminal (residues 621-732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650-697 of HSP90alpha were essential for the binding to the middle domain. An almost identical region (residues 657-720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662-678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90alpha and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins.