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

The α isoform of human 90‐kDa heat shock protein (HSP90α) 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 HSP90α 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 HSP90α 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.