Kinetic analysis of interdomain coupling in a lidless variant of the molecular chaperone DnaK: DnaK's lid inhibits transition to the low affinity state.
Kinetic analysis of interdomain coupling in a lidless variant of the molecular chaperone DnaK: DnaK's lid inhibits transition to the low affinity state.
复制标题
分子伴侣 DnaK 的无盖变体中域间耦合的动力学分析：DnaK 的盖子抑制向低亲和力状态的转变。
DOI：
10.1021/bi0263208
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Witt,StephanN
中科院分区：
文献类型：
--
作者：
Slepenkov,SergeyV;Witt,StephanN
DnaK, theEscherichia coliHsp70, possesses two functional domains, the N- and C-terminal ATPase and peptide-binding domains, respectively. Elucidation of the mechanism of allosteric coupling between the two domains is key to understanding how Hsp70 chaperones interact with their substrates. We previously reported that ATP reacts with wild-type DnaK−peptide complexes according to the two-step reaction, ATP + DnaK−P ⇔ ATP−DnaK−P ⇔ ATP−DnaK* + P, where ATP binds in the first step, and a conformational change that quenches DnaK's tryptophan fluorescence (denoted by the asterisk) and expels bound peptide occurs in the second step. Here we report that DnaK(2-517), a lidless variant, also reacts with ATP and peptide by this two-step mechanism. Compared to wild-type DnaK, we found that, depending on the sequence of the bound peptide and the temperature, deletion of the lid produces a 27- to 66-fold increase in the rate constant (k2) for the ATP-triggered conformational change (ATP−DnaK−P → ATP−DnaK*+P) but only a ∼2-fold increase in the rate constant (k-2) for the reverse reaction (ATP−DnaK*+P → ATP−DnaK−P). A model is proposed in which the lid regulates the rate of interdomain communication by retarding motions within the β-sandwich that occur as a consequence of ATP binding. New evidence in support of the reversible, two-step conformational switch mechanism is also presented.
