ATP-induced conformational changes in Hsp70: molecular dynamics and experimental validation of an in silico predicted conformation.
ATP-induced conformational changes in Hsp70: molecular dynamics and experimental validation of an in silico predicted conformation.
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DOI：
10.1021/bi901256y
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发表时间：
2009-12-08
期刊：
影响因子：
2.9
通讯作者：
Sousa, Rui
中科院分区：
文献类型：
--
作者：
Woo, Hyung-June;Jiang, Jianwen;Lafer, Eileen M.;Sousa, Rui
70-kD热休克蛋白（Hsp70s）分别通过ATP结合和水解与蛋白质底物释放和结合，在防止蛋白质错误折叠和修复应激损伤中发挥重要作用。ATP结合被认为可以诱导核苷酸周围Hsp70核苷酸结合域（NBD）的关闭。我们在这里报告了对这种开闭过渡的计算/实验联合研究。对分离开态nbd进行了全原子分子动力学模拟。无核苷酸的NBD样品具有广泛的开放构型，表现出其四个子结构域（IA-IIB）的灵活重排。相比之下，atp结合的Hsp70 NBD接近于一系列构型，比在atp配合的NBD晶体中观察到的构型要封闭得多。模拟中观察到的IB和IIB亚结构域的接近导致IB的荧光探针Trp90与IIB的Arg261有很强的配位，这是晶体结构中未见的特征。为了确定这种计算观察到的构象是否发生在溶液中，我们构建了一个R261A突变体。发现该突变增加了ATP的Km和kcat，并显著降低了ATP结合时观察到的荧光猝灭的程度。因此，我们的结果解释了在分离的NBD中观察到的先前无法解释的atp驱动的Trp90荧光变化。
The 70-kD heat shock proteins (Hsp70s) play important roles in preventing the misfolding of proteins and repairing damage under stress by coupling ATP binding and hydrolysis to protein substrate release and binding, respectively. ATP binding is believed to induce closing of the Hsp70 nucleotide binding domain (NBD) around the nucleotide. We report here a combined computational/experimental study of this open-closed transition. All-atom molecular dynamics simulations were performed for isolated open state NBDs with and without bound ATP. The nucleotide-free NBD samples a wide range of open configurations exhibiting flexible rearrangements of its four subdomains (IA-IIB). In contrast, the ATP-bound Hsp70 NBD closes to a range of configurations that is substantially more closed than the conformation observed in crystals of ATP-complexed NBDs. The close approach of subdomains IB and IIB observed in the simulations results in a strong coordination of the fluorescence probe Trp90 of IB with Arg261 of IIB, a feature not seen in the crystal structures. To determine if this computationally observed conformation occurs in solution, we constructed an R261A mutant. The mutation was found to increase the Km and kcat for ATP and to significantly reduce the extent of the fluorescence quench observed upon ATP binding. Our results thus account for the previously unexplained ATP-driven change in Trp90 fluorescence seen in the isolated NBD.
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