Stepwise assembly of a glucocorticoid receptor center dot hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket

A system of five purified proteins that assembles stable glucocorticoid rec eptor (GR)-hsp90 heterocomplexes has been reconstituted from reticulocyte l ysate. Two proteins, hsp90 and hsp70, are required for the activation of st eroid binding activity that occurs with heterocomplex assembly, and three p roteins, Hop, hsp40, p23, act as co-chaperones that enhance activation and assembly (Morishima, Y., Kanelakis, K. C., Silverstein, it RI., Dittmar, IL D., Estrada, L., and Pratt, W. B. (2000) J. Biol, Chem. 275, 6894-6900). H ere we demonstrate that the first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR After elimination of fr ee hsp70, these preformed GR.hsp70 complexes can be activated to the steroi d binding state by the hsp70 free assembly system in a second ATP-dependent step. hsp90 is required for opening of the steroid binding pocket and is c onverted to its ATP-dependent conformation during this second step. We pred ict that hsp70 in its ATP-dependent conformation binds initially to the fol ded receptor and is then converted to the ADP-dependent form with high affi nity for hydrophobic substrate. This conversion initiates the opening of th e hydrophobic steroid binding pocket such that it can now accept the hydrop hobic binding form of hsp90, which in turn must be converted to its ATP-dep endent conformation for the pocket to be accessible by steroid.