Popp Simone, Packschies Lars, Radzwill Nicole, Vogel Klaus Peter, Steinhoff Heinz-Jürgen, Reinstein Jochen
Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany.
J Mol Biol. 2005 Apr 15;347(5):1039-52. doi: 10.1016/j.jmb.2005.02.026.
The molecular chaperone DnaK recognizes and binds substrate proteins via a stretch of seven amino acid residues that is usually only exposed in unfolded proteins. The binding kinetics are regulated by the nucleotide state of DnaK, which alternates between DnaK.ATP (fast exchange) and DnaK.ADP (slow exchange). These two forms cycle with a rate mainly determined by the ATPase activity of DnaK and nucleotide exchange. The different substrate binding properties of DnaK are mainly attributed to changes of the position and mobility of a helical region in the C-terminal peptide-binding domain, the so-called LID. It closes the peptide-binding pocket and thus makes peptide binding less dynamic in the ADP-bound state, but does not (strongly) interact with peptides directly. Here, we address the question if nucleotide-dependent structural changes may be observed in the peptide-binding region that could also be connected to peptide binding kinetics and more importantly could induce structural changes in peptide stretches using the energy available from ATP hydrolysis. Model peptides containing two cysteine residues at varying positions were derived from the structurally well-documented peptide NRLLLTG and labelled with electron spin sensitive probes. Measurements of distances and mobilities of these spin labels by electron paramagnetic resonance spectroscopy (EPR) of free peptides or peptides bound to the ATP and ADP-state of DnaK, respectively, showed no significant changes of mobility nor distance of the two labels. This indicates that no structural changes that could be sensed by the probes at the position of central leucine residues located in the center of the binding region occur due to different nucleotide states. We conclude from these studies that the ATPase activity of DnaK is not connected to structural changes of the peptide-binding pocket but rather only has an effect on the LID domain or other further remote residues.
分子伴侣DnaK通过一段通常仅在未折叠蛋白中暴露的七个氨基酸残基序列识别并结合底物蛋白。结合动力学受DnaK核苷酸状态的调节，DnaK的核苷酸状态在DnaK.ATP（快速交换）和DnaK.ADP（慢速交换）之间交替。这两种形式的循环速率主要由DnaK的ATP酶活性和核苷酸交换决定。DnaK不同的底物结合特性主要归因于C端肽结合结构域中一个螺旋区域（即所谓的LID）的位置和流动性变化。它关闭了肽结合口袋，因此在ADP结合状态下使肽结合的动态性降低，但不直接与肽（强烈）相互作用。在这里，我们探讨是否可以在肽结合区域观察到核苷酸依赖性结构变化，这种变化也可能与肽结合动力学相关，更重要的是，是否可以利用ATP水解产生的能量诱导肽段发生结构变化。从结构上已充分证明的肽NRLLLTG衍生出在不同位置含有两个半胱氨酸残基的模型肽，并用电子自旋敏感探针进行标记。分别通过游离肽或与DnaK的ATP和ADP状态结合的肽的电子顺磁共振光谱（EPR）测量这些自旋标记的距离和流动性，结果表明两个标记的流动性和距离均无显著变化。这表明，由于核苷酸状态不同，位于结合区域中心的中央亮氨酸残基位置处未发生可被探针检测到的结构变化。我们从这些研究中得出结论，DnaK的ATP酶活性与肽结合口袋的结构变化无关，而仅对LID结构域或其他更远端的残基有影响。
