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

Yoshihiro Morishima,William B Pratt,Da-Pei Li,Patrick J.M Murphy,Edwin R Sanchez

doi:10.1074/jbc.m000434200

Abstract

A system of five purified proteins that assembles stable glucocorticoid receptor (GR)-hsp90 heterocomplexes has been reconstituted from reticulocyte lysate. Two proteins, hsp90 and hsp70, are required for the activation of steroid binding activity that occurs with heterocomplex assembly, and three proteins, Hop, hsp40, p23, act as co-chaperones that enhance activation and assembly (Morishima, Y., Kanelakis, K. C., Silverstein, A.M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900). Here 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 free hsp70, these preformed GR.hsp70 complexes can be activated to the steroid 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 converted to its ATP-dependent conformation during this second step. We predict that hsp70 in its ATP-dependent conformation binds initially to the folded receptor and is then converted to the ADP-dependent form with high affinity for hydrophobic substrate. This conversion initiates the opening of the hydrophobic steroid binding pocket such that it can now accept the hydrophobic binding form of hsp90, which in turn must be converted to its ATP-dependent conformation for the pocket to be accessible by steroid.

Highlights

Unliganded steroid receptors exist in cytosols in heterocomplexes with the abundant, ubiquitous, and essential heat shock protein hsp901
glucocorticoid receptor (GR) immune pellets stripped of insect hsp90 were incubated at 30 °C under the buffer conditions required for heterocomplex assembly
All of the receptors in L cell cytosol are bound to hsp90, and 75–100% of the stripped receptors are reactivated by reticulocyte lysate to the steroid binding state [23]

Summary

Introduction

Unliganded steroid receptors exist in cytosols in heterocomplexes with the abundant, ubiquitous, and essential heat shock protein hsp901 (for review, see Ref. 1). Two of these proteins, hsp and hsp, are absolutely required for opening the steroid binding cleft in the GR LBD, and the other three proteins act as co-chaperones that increase the overall efficiency of GR1⁄7hsp heterocomplex assembly [17]. Hop is not required for opening of the steroid binding cleft in the GR LBD, it increases the rate of the process [17]. The ADP-bound conformation of hsp has a high affinity for hydrophobic substrates, and hsp (provided as the purified yeast homolog YDJ-1) increases GR1⁄7hsp heterocomplex assembly [13] but it is not required for assembly [17]. To have an open steroid binding cleft, the receptor-bound hsp must assume its ATP-dependent conformation [22], which is stabilized by p23 [12]

Methods

Results

Conclusion