Dissecting the role of a conserved motif (the second region of homology) in the AAA family of ATPases - Site-directed mutagenesis of the ATP-dependent protease FtsH
Dissecting the role of a conserved motif (the second region of homology) in the AAA family of ATPases - Site-directed mutagenesis of the ATP-dependent protease FtsH
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DOI：
10.1074/jbc.274.37.26225
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发表时间：
1999-09-10
影响因子：
4.8
通讯作者：
Ogura, T
中科院分区：
文献类型：
--
作者：
Karata, K;Inagawa, T;Ogura, T
大肠杆菌FtsH是一种ATP依赖性蛋白酶，属于AAA蛋白家族。第二同源区（SRH）是AAA家族成员中高度保守的基序，并将这些蛋白质与更广泛的Walker型ATP酶家族部分区分开来。尽管它在AAA蛋白家族中保守，但关于SRH的功能知之甚少。为了解决这个问题，我们引入点突变系统到SRH的FtsH和研究的突变蛋白的活动。发现SRH内高度保守的氨基酸残基对于FtsH的功能是关键的，在这些位置的突变导致ATP酶活性降低或消除。突变对FtsH蛋白酶活性的影响与其对ATP酶活性的影响显著相关。ATP酶缺陷的SRH突变体进行了ATP诱导的构象变化类似于野生型FtsH，这表明了重要的作用，SRH在ATP水解，但不ATP结合。根据N-乙基马来酰亚胺敏感性融合蛋白的六聚化结构域的晶体结构的数据分析表明，AAA ATP酶的ATP水解的合理机制，其引起SRH的分子间催化作用。
Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins. Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH.