Briknarová K, Takayama S, Brive L, Havert M L, Knee D A, Velasco J, Homma S, Cabezas E, Stuart J, Hoyt D W, Satterthwait A C, Llinás M, Reed J C, Ely K R
The Burnham Institute, La Jolla, California 92037, USA.
Nat Struct Biol. 2001 Apr;8(4):349-52. doi: 10.1038/86236.
BAG-family proteins share a conserved protein interaction region, called the 'BAG domain', which binds and regulates Hsp70/Hsc70 molecular chaperones. This family of cochaperones functionally regulates signal transducing proteins and transcription factors important for cell stress responses, apoptosis, proliferation, cell migration and hormone action. Aberrant overexpression of the founding member of this family, BAG1, occurs in human cancers. In this study, a structure-based approach was used to identify interacting residues in a BAG1--Hsc70 complex. An Hsc70-binding fragment of BAG1 was shown by multidimensional NMR methods to consist of an antiparallel three-helix bundle. NMR chemical shift experiments marked surface residues on the second (alpha 2) and third (alpha 3) helices in the BAG domain that are involved in chaperone binding. Structural predictions were confirmed by site-directed mutagenesis of these residues, resulting in loss of binding of BAG1 to Hsc70 in vitro and in cells. Molecular docking of BAG1 to Hsc70 and mutagenesis of Hsc70 marked the molecular surface of the ATPase domain necessary for interaction with BAG1. The results provide a structural basis for understanding the mechanism by which BAG proteins link molecular chaperones and cell signaling pathways.
BAG家族蛋白共享一个保守的蛋白质相互作用区域，称为“BAG结构域”，该结构域可结合并调节Hsp70/Hsc70分子伴侣。这一同伴分子伴侣家族在功能上调节对细胞应激反应、细胞凋亡、增殖、细胞迁移和激素作用至关重要的信号转导蛋白和转录因子。该家族的创始成员BAG1在人类癌症中异常过表达。在本研究中，采用基于结构的方法来确定BAG1-Hsc70复合物中的相互作用残基。通过多维核磁共振方法表明，BAG1的一个Hsc70结合片段由一个反平行的三螺旋束组成。核磁共振化学位移实验标记了BAG结构域中第二（α2）和第三（α3）螺旋上参与伴侣结合的表面残基。通过对这些残基进行定点诱变证实了结构预测，导致BAG1在体外和细胞中与Hsc70的结合丧失。BAG1与Hsc70的分子对接以及Hsc70的诱变标记了与BAG1相互作用所需的ATP酶结构域的分子表面。这些结果为理解BAG蛋白连接分子伴侣和细胞信号通路的机制提供了结构基础。
