The Region Adjacent to the Highly Immunogenic Site and Shielded by the Middle Domain Is Responsible for Self-Oligomerization/Client Binding of the HSP90 Molecular Chaperone

Abstract

We here investigated the mechanism of self-oligomerization of the 90-kDa heat shock protein (HSP90) molecular chaperone, because it is known that this oligomerization reflects the client-binding activity. The transition temperatures for the self-oligomerization of the full-length forms of human HSP90alpha and HtpG (bacterial HSP90), i.e., 45 and 60 degrees C, respectively, were identical to those for the dissociation of the recombinant N domain (residues 1-400 of human HSP90alpha and residues 1-336 of HtpG in our definition) from the remainder of the molecule. The N domain of human HSP90alpha expressed in Escherichia coli was oligomeric, and the oligomerization activity was localized within residues 311-350, i.e., C-terminally adjacent to the highly immunogenic site (residues 291-304). Particularly, residues 341-350 were critical on oligomerization. On the other hand, residues 289-389 were indispensable for the interaction with the M domain (residues 401-618) of the molecule. Oligomer formation of the N domain was efficiently suppressed by its extension until Lys546, i.e., residues 401-546, which is required for the interaction with the N domain. Among highly conserved amino acids at residues 289-400, Trp297, Pro379, and Phe384 were essential for the interaction with the M domain. With these observations taken together, we propose as the activation mechanism of HSP90 molecular chaperone that heat stress induces the liberation of the oligomerization/client-binding site of residues 311-350 by disrupting the intramolecular interaction between residues 289-389 and 401-546.

Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli

“…His 6 ‐tagged recombinant proteins were expressed and purified as described previously [33]. Briefly, Y1090[pREP4] carrying pQE9‐ or pQE60‐derived expression plasmids was cultured in LB broth containing 50 μg·mL −1 of ampicillin and 25 μg·mL −1 of kanamycin at 37 °C overnight.…”

Identification of the pentapeptide constituting a dominant epitope common to all eukaryotic heat shock protein 90 molecular chaperones

“…This discusbinding site (Meyer et al 2003). We recently mapped amino acids 311-350 as an essential region for self-oligomerization/client binding (Nemoto et al 2004). Moreover, a similar ␤-sheet platform is involved in client binding of DnaK (Zhu et al 1996) and major histocompatibility complex-class I molecule (Fremont et al 1995).…”

An Escherichia coli expression system for glutamyl endopeptidases optimized by complete suppression of autodegradation

“…Recombinant proteins were expressed and purified as described previously [16]. Briefly, E. coli Y1090[pREP4] (300 ml) carrying pQE60-derived expression plasmids was cultured overnight at 37˚C…”