Mechanism of DnaB helicase of Escherichia coli:: Structural domains involved in ATP hydrolysis, DNA binding, and oligomerization
Mechanism of DnaB helicase of Escherichia coli:: Structural domains involved in ATP hydrolysis, DNA binding, and oligomerization
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DOI：
10.1021/bi990048t
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发表时间：
1999-08-24
期刊：
影响因子：
2.9
通讯作者：
Biswas, SB
中科院分区：
文献类型：
--
作者：
Biswas, EE;Biswas, SB
我们描述了大肠杆菌DNAB解旋酶的三个不同的结构域：a区，氨基酸残基(AA)1-156；β区，a157-302；和区Gamma，aa303-471。利用这些区域缺失的突变体，我们研究了它们在六聚体形成、DNA依赖的ATPase和DNA解旋酶活性中的作用(S)。突变的DNABβ伽马蛋白，其中结构域α被删除，形成一个六聚体；而突变的DNABα蛋白，其中的伽马结构域被删除，只能形成二聚体。DNABαβ的二聚化是依赖于镁离子的。这些数据表明，DNAB解旋酶的寡聚至少涉及两个不同的蛋白质-蛋白质相互作用位点；其中一个主要位于β结构域(第1位)，而另一个相互作用位点位于伽马结构域(第2位)。突变的DNABβ是一种147aa的多肽，其中a和Gamma结构域都被缺失，显示出完全功能的ATPase活性。因此，这个结构域构成了ATPase活性的“中心催化结构域”。DNABαβ和DNABβ的ATPase活性在动力学上是相似的，表明结构域α对ATPase活性影响很小或没有影响。在这两种情况下，ATPase活性都不依赖于DNA。DNABβ-Gamma具有DNA依赖的ATPase活性，与野生型DNAB蛋白(WtDna B)的ATPase活性在动力学上相当，表明C-末端结构域Gamma在增强结构域β的ATPase活性以及DNA结合方面具有独特的作用。缺失结构域or的突变体DNABβ-Gamma没有任何解旋酶活性，表明结构域cc具有重要作用。这项研究的主要发现是：(I)结构域β包含一个功能性的ATPase活性部位；(Ii)结构域Gamma似乎是结构域的DNA结合结构域和ATPase活性的正调节因子；(Iii)尽管结构域或结构域对ATPase、DNA结合活性或六聚体的形成没有任何显著影响，但它在将ATP水解的能量传递给六聚体解离DNA的过程中肯定起着关键作用；以及(Iv)这三个结构域都是解旋酶活性所必需的。
We describe the delineation of three distinct structural domains of the DnaB helicase of Escherichia coli: domain a, amino acid residues (aa) 1-156; domain beta, aa 157-302; and domain gamma, aa 303-471. Using mutants with deletion in these domains, we have examined their role(s) in hexamer formation, DNA-dependent ATPase, and DNA helicase activities. The mutant DnaB beta gamma protein, in which domain alpha was deleted, formed a hexamer; whereas the mutant DnaB alpha beta, in which domain gamma was deleted, could form only dimers. The dimerization of DnaB alpha beta was Mg2+ dependent. These data suggest that the oligomerization of DnaB helicase involves at least two distinct protein-protein interaction sites; one of these sites is located primarily within domain beta (site 1), while the other interaction site is located within domain gamma (site 2). The mutant DnaB beta, a polypeptide of 147 aa, where both domains a and gamma were deleted, displayed a completely functional ATPase activity. This domain, thus, constitutes the "central catalytic domain" for ATPase activity. The ATPase activity of DnaB alpha beta was kinetically comparable to that of DnaB beta, indicating that domain alpha had little or no influence on the ATPase activity. In both cases, the ATPase activities were DNA independent. DnaB beta gamma had a DNA-dependent ATPase activity that was kinetically comparable to the ATPase activity of wild-type DnaB protein (wtDnaB), indicating a specific role for C-terminal domain gamma in enhancement of the ATPase activity of domain beta as well as in DNA binding. Mutant DnaB beta gamma, which lacked domain or, was devoid of any helicase activity pointing to a significant role for domain cc. The major findings of this study are (i) domain beta contained a functional ATPase active site; (ii) domain gamma appeared to be the DNA binding domain and a positive regulator of the ATPase activity of domain beta; (iii) although domain or did not have any significant effect on the ATPase, DNA binding activities, or hexamer formation, it definitely plays a pivotal role in transducing the energy of ATP hydrolysis to DNA unwinding by the hexamer; and (iv) all three domains are required for helicase activity.