Structural organization of the hsp90 /hsp70 -based chaperone machinery and its interaction with the glucocorticoid receptor ligand binding domain

Five proteins---hsp90, hsp70, Hop, hsp40, and p23---constitute a multiprotein chaperone system that assembles the glucocorticoid receptor (GR) into a complex with hsp90 and converts the ligand binding domain (LBD) of the receptor to its high affinity binding state. The GR is first primed by hsp70/hsp40, and subsequently reactivated by hsp90, with the GR•hsp90 heterocomplex being stabilized by the hsp90 co-chaperone p23.;This thesis presents the stoichiometry, abundance, and functional significance of the hsp90/hsp70-based chaperone machinery in reticulocyte lysate. All of the Hop in reticulocyte lysate is present in an hsp90•Hop•hsp70 complex with a stoichiometry of 2:1:1. The complex accounts for ∼30% of the hsp90 and ∼9% of the hsp70 in the lysate, and it possesses the lysate's GR•hsp90 heterocomplex assembly activity. Consistent with the notion that hsp90 and hsp70 cooperate with each other to open the steroid binding cleft, purified hsp90 and hsp70 interact directly to form a weak hsp90•hsp70 complex with a stoichiometry of 2:1.;This thesis also demonstrates that hsp90 binding to the receptor requires the presence, but not defined composition, of a seven-amino acid sequence at the amino terminus of the GR LBD. A GR construct containing three point mutations in this seven-amino acid sequence displays reduced binding of steroid and hsp90 in whole cells but wild type affinity for steroid and normal hsp90 binding under cell-free conditions. These properties are consistent with an increased rate of mutant GR•hsp90 heterocomplex disassembly in the cell. Mutation of the entire seven-amino acid sequence disconnects hsp90 binding from opening of the ligand binding cleft and steroid binding activity.;Finally, this thesis describes the visualization and assembly of the primed GR•hsp70 complex. It shows the most common stoichiometry of the GR•hsp70 complex to be 1:1 with some complexes 1:2 and a few of larger size. In contrast to recent observations with the progesterone receptor, hsp40 does not target hsp70 to the GR; rather, hsp40 functions as an hsp70 co-chaperone and is active at substoichiometric levels. A GR LBD amino-terminal truncation mutant lacking steroid binding activity is nonetheless able to be primed by hsp70, indicating it is the subsequent assembly step with hsp90 that is defective.