Sewell B Trevor, Best Robert B, Chen Shaoxia, Roseman Alan M, Farr George W, Horwich Arthur L, Saibil Helen R
Electron Microscope Unit and Department of Chemistry, University of Cape Town, Rondebosch, South Africa.
Nat Struct Mol Biol. 2004 Nov;11(11):1128-33. doi: 10.1038/nsmb844. Epub 2004 Oct 10.
The chaperonin GroEL assists protein folding through ATP-dependent, cooperative movements that alternately create folding chambers in its two rings. The substitution E461K at the interface between these two rings causes temperature-sensitive, defective protein folding in Escherichia coli. To understand the molecular defect, we have examined the mutant chaperonin by cryo-EM. The normal out-of-register alignment of contacts between subunits of opposing wild-type rings is changed in E461K to an in-register one. This is associated with loss of cooperativity in ATP binding and hydrolysis. Consistent with the loss of negative cooperativity between rings, the cochaperonin GroES binds simultaneously to both E461K rings. These GroES-bound structures were unstable at higher temperature, dissociating into complexes of single E461K rings associated with GroES. Lacking the allosteric signal from the opposite ring, these complexes cannot release their GroES and become trapped, dead-end states.
伴侣蛋白GroEL通过依赖ATP的协同运动协助蛋白质折叠，这种运动在其两个环中交替形成折叠腔。这两个环之间界面处的E461K替换导致大肠杆菌中对温度敏感的、有缺陷的蛋白质折叠。为了理解分子缺陷，我们通过冷冻电镜检查了突变伴侣蛋白。在E461K中，相对野生型环的亚基之间正常的错位接触排列变为了对齐排列。这与ATP结合和水解中的协同性丧失有关。与环之间负协同性的丧失一致，共伴侣蛋白GroES同时结合到两个E461K环上。这些与GroES结合的结构在较高温度下不稳定，解离成与GroES相关的单个E461K环的复合物。由于缺乏来自相对环的变构信号，这些复合物无法释放它们的GroES并陷入死胡同状态。
