Xu M, Dittmar K D, Giannoukos G, Pratt W B, Simons S S
Steroid Hormones Section, NIDDK/LMCB, National Institutes of Health, Bethesda, Maryland 20892-0805, USA.
J Biol Chem. 1998 May 29;273(22):13918-24. doi: 10.1074/jbc.273.22.13918.
The glucocorticoid receptor (GR) HBD must be bound to the protein chaperone hsp90 in order to acquire the high affinity steroid binding conformation. Despite this crucial role of hsp90, its binding site in GR remains poorly defined. Large portions of the GR HBD have been implicated and no similarity has been established between steroid receptor HBDs and the catalytic domains of the protein kinases (e.g. pp60(src), Raf) that also form stable heterocomplexes with hsp90. Thus, it has been thought that some general property of the proteins, such as exposure of hydrophobic residues in partially denatured regions, determines the assembly of stable hsp90 heterocomplexes. In this work, we have studied fusion proteins containing glutathione S-transferase (GST) and very short amino-terminal truncations just before and at the beginning of the rat GR HBD that are otherwise intact to the carboxyl terminus. Overexpression in COS cells of the chimeras GST537C and GST547C was found to yield receptors that were bound to hsp90 and had wild-type steroid binding affinity. However, removal of 7 more amino acids to form GST554C resulted in a fusion protein that did not bind either hsp90 or steroid. Additional mutations revealed that the role of these 7 amino acids was neither to provide a spacer between protein domains nor to expose a protein surface by introducing a bend in the conserved alpha-helix. Instead, these observations support a model in which the sequence of the 7 amino acids directly or indirectly affects hsp90 binding to the GR HBD. Thus, a region of GR that has not been thought to be relevant for hsp90 binding is now seen to be of critical importance, and these data argue strongly against the commonly accepted model of receptor-hsp90 heterocomplex assembly in which the chaperone initially interacts nonspecifically with hydrophobic regions of the partially denatured HBD and subsequently assists its folding to the steroid binding confirmation.
糖皮质激素受体（GR）的激素结合结构域（HBD）必须与蛋白伴侣hsp90结合，才能获得高亲和力的类固醇结合构象。尽管hsp90发挥着这一关键作用，但其在GR中的结合位点仍不清楚。GR HBD的大部分区域都被认为与该作用有关，而且类固醇受体HBD与同样能和hsp90形成稳定异源复合物的蛋白激酶（如pp60(src)、Raf）的催化结构域之间未发现相似性。因此，人们认为蛋白质的一些普遍特性，如部分变性区域中疏水残基的暴露，决定了稳定的hsp90异源复合物的组装。在这项研究中，我们研究了含有谷胱甘肽S-转移酶（GST）的融合蛋白，这些融合蛋白在大鼠GR HBD之前及起始处有非常短的氨基末端截短，而其羧基末端则保持完整。发现嵌合体GST537C和GST547C在COS细胞中的过表达产生了与hsp90结合且具有野生型类固醇结合亲和力的受体。然而，再去除7个氨基酸形成GST554C后，得到的融合蛋白既不与hsp90结合，也不与类固醇结合。进一步的突变表明，这7个氨基酸的作用既不是在蛋白结构域之间提供间隔，也不是通过在保守的α螺旋中引入弯曲来暴露蛋白质表面。相反，这些观察结果支持了一种模型，即这7个氨基酸的序列直接或间接影响hsp90与GR HBD的结合。因此，GR中一个以前认为与hsp90结合无关的区域现在被认为至关重要，而且这些数据有力地反驳了普遍接受的受体-hsp90异源复合物组装模型，该模型认为伴侣蛋白最初与部分变性的HBD的疏水区域非特异性相互作用，随后协助其折叠成类固醇结合构象。
