Receiver Domains Control the Active-State Stoichiometry of Aquifex aeolicus σ54 Activator NtrC4, as Revealed by Electrospray Ionization Mass Spectrometry
Receiver Domains Control the Active-State Stoichiometry of Aquifex aeolicus σ54 Activator NtrC4, as Revealed by Electrospray Ionization Mass Spectrometry
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DOI：
10.1016/j.jmb.2009.08.033
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发表时间：
2009-10-30
影响因子：
5.6
通讯作者：
Wemmer, David E.
中科院分区：
文献类型：
--
作者：
Batchelor, Joseph D.;Sterling, Harry J.;Wemmer, David E.
蛋白质体外研究的一个共同挑战是确定哪些结构和条件最具生理学意义。Sigma(54)激活剂是一种蛋白质，经过调节组装后形成一个活性的ATPase环，使Sigma(54)聚合酶能够转录。以前对Sigma(54)激活子的AAA(+)ATPase结构域的研究表明，一些是七聚体，另一些是六聚体。因为活性寡聚体是从关闭状态的二聚体组装而来的，所以人们认为偶数个寡聚体应该占主导地位，当研究激活剂的单个结构域而不是完整的蛋白质时，七聚体的形成就会发生。在这里，我们介绍了电喷雾电离质谱学实验的结果，表征了完整的NtrC4(极端嗜热者Aquifex aeolicus的sigma(54)激活剂)的组装状态，以及它的ATPase结构域，以及调节-ATPase和ATPase-DNA结合结构域的组合。我们发现，全长和激活的调节性ATPase蛋白形成六聚体，而分离的ATPase结构域、未激活的调节性ATPase和ATPase-DNA结合结构域形成七聚体。相对于七聚体，N-末端调节域的激活是稳定ATPase六聚体形式的关键因素。(C)2009爱思唯尔有限公司。保留所有权利。
A common challenge with studies of proteins in vitro is determining which constructs and conditions are most physiologically relevant. sigma(54) activators are proteins that undergo regulated assembly to form an active ATPase ring that enables transcription by sigma(54)-polymerase. Previous studies of AAA(+) ATPase domains from sigma(54) activators have shown that some are heptamers, while others are hexamers. Because active oligomers assemble from off-state dimers, it was thought that even-numbered oligomers should dominate, and that heptamer formation would occur when individual domains of the activators, rather than the intact proteins, were studied. Here we present results from electrospray ionization mass spectrometry experiments characterizing the assembly states of intact NtrC4 (a sigma(54) activator from Aquifex aeolicus, an extreme thermophile), as well as its ATPase domain alone, and regulatory-ATPase and ATPase-DNA binding domain combinations. We show that the full-length and activated regulatory-ATPase proteins form hexamers, whereas the isolated ATPase domain, unactivated regulatory-ATPase, and ATPase-DNA binding domain form heptamers. Activation of the N-terminal regulatory domain is the key factor stabilizing the hexamer form of the ATPase, relative to the heptamer. (c) 2009 Elsevier Ltd. All rights reserved.