GrpE accelerates nucleotide exchange of the molecular chaperone DnaK with an associative displacement mechanism
GrpE accelerates nucleotide exchange of the molecular chaperone DnaK with an associative displacement mechanism
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DOI：
10.1021/bi962835l
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发表时间：
1997-03-25
期刊：
影响因子：
2.9
通讯作者：
Reinstein, J
中科院分区：
文献类型：
--
作者：
Packschies, L;Theyssen, H;Reinstein, J
分子伴侣DNAK的ATP水解性和蛋白结合释放循环受辅助蛋白GRPE和DNAJ的调节。本文通过平衡实验和停流动力学实验研究了分子伴侣DNAK及其核苷酸交换因子GRPE与荧光二磷酸腺苷类似物5‘-二磷酸(N-8-[4-[(N’-methylanthraniloyl)amino]butyl]-8-aminoadenosine-ADP)之间形成的络合物。GRPE刺激的核苷酸交换的催化循环涉及三元DNAK。GRPE。ADP复合体和二元DNAK。GRPE和DNAK。ADP复合体。GRPE与DNAK、ADP和无核苷酸DNAK相互作用的平衡数据可以用一个简单的平衡系统来描述，其中GRPE将ADP与DNAK的亲和力降低了200倍。然而，瞬时动力学研究表明，GRPE的功能循环还包括至少两个不同的三元DNAK GRPE ADP络合物。结果表明，GRPE与DNAK-ADP的初始弱结合后，三元络合物的异构化导致核苷酸结合减弱，最后迅速解离，GRPE对核苷酸交换的最大刺激倍数为5000倍。我们认为，这种动力学观察到的异构化代表着DNAK的核苷酸结合口袋的结构变化(打开)，从而允许快速的核苷酸交换。
The ATP hydrolysis and protein-binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ. Here we describe a study of the formation of complexes between the molecular chaperone DnaK, its nucleotide exchange factor GrpE, and the fluorescent ADP analog N-8-[4-[(N'-methylanthraniloyl)amino]butyl]-8-aminoadenosine 5'-diphosphate (MABA-ADP) by equilibrium and stopped flow kinetic experiments. The catalytic cycle of the GrpE-stimulated nucleotide exchange involves a ternary DnaK . GrpE . ADP complex as well as the binary DnaK . GrpE and DnaK . ADP complexes. The equilibrium data of the interaction of GrpE with DnaK ADP and the nucleotide-free DnaK can be described by a simple equilibrium system where GrpE reduces the affinity of ADP for DnaK 200-fold. However, transient kinetic studies revealed that the functional cycle of GrpE in addition includes at least two distinct ternary DnaK GrpE ADP complexes. Our data indicate that the initial weak binding of GrpE to DnaK ADP is followed by an isomerization of the ternary complex which leads to weakening of nucleotide binding and finally to its rapid dissociation, The maximal stimulation for nucleotide exchange brought about by GrpE was found to be 5000-fold. We propose that this kinetically observed isomerization represents a structural change (opening) of the nucleotide binding pocket of DnaK that allows for fast nucleotide exchange.