Mechanism of Dimer Formation of the 90‐kDa Heat‐Shock Protein

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Abstract
This study describes the mechanism of homodimer formation of the 90-kDa heat-shock protein (HSP90). In eukaryotic cells, there are two HSP90 isoforms, α and β, encoded by two separate genes. HSP90α exists predominantly as a homodimer, HSP90β mainly as a monomer. Analysis by native PAGE revealed that bacterially expressed HSP90α fused to glutathione S-transferase (GST) existed as a high-molecular-mass oligomer, and was converted to a homodimer following removal of the fusion enzyme by thrombin cleavage. A deletion mutant, HSP90αD44–603, formed a monomer and an N-terminal truncated mutant, HSP90α533–732, existed as a dimer, indicating that the dimer-forming ability resides somewhere in the C-terminal 200 amino acids. Limited proteolysis of the C-terminal 200 amino acids of HSP90α with chymotrypsin produced the C-terminal 16-kDa fragment (Met628/Ala629–Asp732) and its adjacent more N-terminal 13-kDa fragment (Val542–Tyr627/Met628). Size-exclusion HPLC and two-dimensional PAGE analyses demonstrated that these two chymotryptic fragments bound each other. The C-terminal 198 amino acids as well as the full-length form of HSP90β revealed a lower dimer-forming activity than HSP90α. Expression of the chimeric proteins at the C-terminal 198 amino acids of the α and β isoforms further indicated that the 16 amino acid substitutions locating between amino acids 561 and 685 account for the impeded dimerization of HSP90β. A leucine zipper motif (Met402–Leu423) was unlikely to be involved in the dimer formation. Taken together, these results indicate that the dimeric structure of HSP90α is mediated by the C-terminal 191 amino acids and consists of duplicate interactions of the C-terminal region (Met628/Ala629–Asp732) of one subunit and the adjacent more N-terminal region (Val542–Try627/Met628) of the other subunit.