Characterization of a lidless form of the molecular chaperone DnaK - Deletion of the lid increases peptide on- and off-rate constants
Characterization of a lidless form of the molecular chaperone DnaK - Deletion of the lid increases peptide on- and off-rate constants
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DOI：
10.1074/jbc.m100237200
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发表时间：
2001-07-20
影响因子：
4.8
通讯作者：
Witt, SN
中科院分区：
文献类型：
--
作者：
Buczynski, G;Slepenkov, SV;Witt, SN
70 kDa 分子伴侣 DnaK 的 C 端多肽结合结构域由独特的盖子状子结构域组成，该子结构域似乎阻碍了肽结合位点的空间访问。我们表达、纯化并表征了无盖形式的 DnaK，以测试盖对 ATP 酶活性、域间通讯以及肽结合动力学的影响。主要发现是，盖子的丢失会产生一种激活形式的 DnaK，它与 ATP 结合的 DnaK 不同。例如，在 25°C 时，NR 肽 (NRLLLTG) 从 DnaK 的 ADP 和 ATP 状态解离，观察到的解离速率常数分别为 0.001 和 4.8 s(-1)。相反，对于缺乏大部分螺旋盖（残基 518-638）的 DnaK，NR 肽以观察到的解离速率常数 0.1 和 188 s(-1) 解离。这些结果表明，盖子的丢失不会干扰域间通讯，β-夹心肽结合结构域可以以两种离散构象存在，并且盖子的功能是增加 DnaK 肽复合物的寿命。我们讨论了几种机制来解释盖子如何影响 DnaK 肽复合物的寿命。
The C-terminal, polypeptide binding domain of the 70-kDa molecular chaperone DnaK is composed of a unique lidlike subdomain that appears to hinder steric access to the peptide binding site. We have expressed, purified, and characterized a lidless form of DnaK to test the influence of the lid on the ATPase activity, on interdomain communication, and on the kinetics of peptide binding. The principal findings are that loss of the lid creates an activated form of DnaK which is not equivalent to ATP-bound DnaK. For example, at 25 degreesC the NR peptide (NRLLLTG) dissociates from the ADP and ATP states of DnaK with observed off-rate constants of 0.001 and 4.8 s(-1), respectively. In contrast, for DnaK that lacks most of the helical lid, residues 518-638, the NR peptide dissociates with observed off-rate constants of 0.1 and 188 s(-1). These results show that the loss of the lid does not interfere with interdomain communication, that the beta -sandwich peptide binding domain can exist in two discrete conformations, and that the lid functions to increase the lifetime of a DnaK peptide complex. We discuss several mechanisms to explain how the lid affects the lifetime of a DnaK peptide complex.