Benaroudj N, Fouchaq B, Ladjimi M M
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette Cedex, France.
J Biol Chem. 1997 Mar 28;272(13):8744-51. doi: 10.1074/jbc.272.13.8744.
We have previously shown that the molecular chaperone HSC70 self-associates in solution into dimers, trimers, and probably high order oligomers, according to a slow temperature- and concentration-dependent equilibrium that is shifted toward the monomer upon binding of ATP peptides or unfolded proteins. To determine the structural basis of HSC70 self-association, the oligomerization properties of the isolated amino- and carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Whereas the amino-terminal ATPase domain (residues 1-384) was found to be monomeric in solution even at high concentrations, the carboxyl-terminal peptide binding domain (residues 385-646) exists as a slow temperature- and concentration-dependent equilibrium involving monomers, dimers, and trimers. The association equilibrium constant obtained for this domain alone is on the order of 10(5) M-1, very close to that determined previously for the entire protein, suggesting that self-association of HSC70 is determined solely by its carboxyl-terminal domain. Furthermore, oligomerization of the isolated carboxyl-terminal peptide binding domain is, like that of the entire protein, reversed by peptide binding, indicating that self-association of the protein may be mediated by the peptide binding site and, as such, should play a role in the regulation of HSC70 chaperone function. A general model for self-association of HSP70 is proposed in which the protein is in equilibrium between two states differing by the conformation of their carboxyl-terminal domain and their self-association properties.
我们之前已经表明，分子伴侣HSC70在溶液中会根据一个缓慢的、温度和浓度依赖性的平衡自缔合形成二聚体、三聚体以及可能的高阶寡聚体，该平衡在结合ATP肽或未折叠蛋白后会向单体方向移动。为了确定HSC70自缔合的结构基础，通过凝胶电泳、尺寸排阻色谱和分析超速离心对该蛋白分离的氨基末端和羧基末端结构域的寡聚化特性进行了分析。虽然发现氨基末端ATP酶结构域（第1至384位氨基酸残基）即使在高浓度下在溶液中也是单体形式，但羧基末端肽结合结构域（第385至646位氨基酸残基）以涉及单体、二聚体和三聚体的缓慢温度和浓度依赖性平衡形式存在。仅针对该结构域获得的缔合平衡常数约为10⁵ M⁻¹，与之前针对整个蛋白测定的结果非常接近，这表明HSC70的自缔合仅由其羧基末端结构域决定。此外，分离的羧基末端肽结合结构域的寡聚化，与整个蛋白一样，会被肽结合逆转，这表明该蛋白的自缔合可能由肽结合位点介导，因此应该在HSC70伴侣功能的调节中发挥作用。提出了一个HSP70自缔合的通用模型，其中该蛋白在两种状态之间处于平衡，这两种状态在其羧基末端结构域的构象及其自缔合特性方面有所不同。
