Protein Associations in DnaA-ATP Hydrolysis Mediated by the Hda-Replicase Clamp Complex*
Protein Associations in DnaA-ATP Hydrolysis Mediated by the Hda-Replicase Clamp Complex*
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DOI：
10.1074/jbc.m412060200
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发表时间：
2005-02
影响因子：
4.8
通讯作者：
M. Su’etsugu;Toh-ru Shimuta;T. Ishida;H. Kawakami;T. Katayama
中科院分区：
文献类型：
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作者：
M. Su’etsugu;Toh-ru Shimuta;T. Ishida;H. Kawakami;T. Katayama
在大肠杆菌中，ATP结合的DnaA蛋白在启动染色体复制中的活性以复制协调的方式受到负控制。RIDA（DnaA调节失活）系统促进DnaA-ATP水解，以产生失活形式的DnaA-ADP，其方式取决于Hda蛋白和β-滑动夹（复制酶全酶的亚基）的DNA加载形式。Hda的一种高功能形式被纯化，并显示在溶液中形成同二聚体，并且发现两个Hda二聚体与单个钳分子结合。纯化的突变体Hda蛋白被用于分阶段的体外RIDA系统，然后通过下拉测定，以显示Hda-钳结合是DnaA-ATP水解的先决条件，并且该结合是由Hda N-末端基序介导的。Hda的AAA+ Box VII基序中的Arg 168在稳定的同源二聚体形成和DnaA-ATP水解中起作用，但在钳夹结合中不起作用。此外，DnaA N-末端结构域是DnaA与Hda-钳复合物的功能性相互作用所必需的。单细胞含有1050个Hda二聚体，与体外实验的结果一致。这些发现和AAA+蛋白（包括DnaA）的特征表明了以下模型。DnaA-ATP在DnaA和Hda的AAA+结构域之间的结合界面处水解; DnaA N-末端结构域支持这种相互作用;并且DnaA-ATP与Hda-夹复合物的相互作用以催化模式发生。
In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the β-sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified mutant Hda proteins were used in a staged in vitro RIDA system followed by a pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arg168 in the AAA+ Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain ∼50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA+ proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA+ domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.