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

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.