Abstract
Heat shock protein 90 (Hsp90) plays a central role in signal transduction and has emerged as a promising target for anti-cancer therapeutics, but its molecular mechanism is poorly understood. At physiological concentration, Hsp90 predominantly forms dimers, but the function of full-length monomers in cells is not clear. Hsp90 contains three domains: the N-terminal and middle domains contribute directly to ATP binding and hydrolysis and the C domain mediates dimerization. To study the function of Hsp90 monomers, we used a single-chain strategy that duplicated the C-terminal dimerization domain. This novel monomerization strategy had the dual effect of stabilizing the C domain to denaturation and hindering intermolecular association of the ATPase domain. The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. The monomeric construct was also defective at ATP hydrolysis and the activation of a kinase and steroid receptor substrate in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric.
Highlights
Among heat shock proteins, Heat shock protein 90 (Hsp90) is unusual because it is not required for the proper folding of most cellular proteins [1] and instead is disproportionately linked to a select group of proteins required for receiving, transducing, and responding to environmental signals
We find that NMCC monomers do not function to support yeast growth, the activation of a kinase nor a hormone receptor substrate in yeast
Our results indicate that dimerization of NMCC Hsp90 is required to position the catalytic machinery for efficient ATP hydrolysis
Summary
Construction of Hsp Variants—All Hsp constructs used in these studies were generated from the yeast HSP82 gene. Samples were prepared in assay buffer with 5 mM dithiothreitol containing 1 M reduced BODIPY-GA and wild-type or NMCC Hsp ranging from 0.1 to 3.3 M These concentrations of Hsp and BODIPY-GA are both well above the 5 nM Kd determined for human Hsp and BODIPY-GA [26]. The frozen cell pellets were lysed by vortexing with glass beads in Src Lysis Buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 0.2 mM sodium orthovanadate to inhibit dephosphorylation, 1 mM phenylmethylsulfonyl fluoride) followed by addition of SDS to 2%. Protein concentration in these SDS lysates was assessed using the BCA assay (Pierce). GR assays were repeated three times starting from fresh yeast colonies
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