Biswas E E, Biswas S B
Department of Molecular Biology, SOM, University of Medicine and Dentistry of New Jersey, Stratford 08084, USA.
Biochemistry. 1999 Aug 24;38(34):10919-28. doi: 10.1021/bi990048t.
We describe the delineation of three distinct structural domains of the DnaB helicase of Escherichia coli: domain alpha, 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 DnaBbetagamma protein, in which domain alpha was deleted, formed a hexamer; whereas the mutant DnaBalphabeta, in which domain gamma was deleted, could form only dimers. The dimerization of DnaBalphabeta was Mg(2+) 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 DnaBbeta, a polypeptide of 147 aa, where both domains alpha 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 DnaBalphabeta was kinetically comparable to that of DnaBbeta, indicating that domain alpha had little or no influence on the ATPase activity. In both cases, the ATPase activities were DNA independent. DnaBbetagamma 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 DnaBbetagamma, which lacked domain alpha, was devoid of any helicase activity pointing to a significant role for domain alpha. 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 alpha 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.
我们描述了大肠杆菌DnaB解旋酶三个不同结构域的划分：α结构域，氨基酸残基（aa）1 - 156；β结构域，aa 157 - 302；γ结构域，aa 303 - 471。利用这些结构域缺失的突变体，我们研究了它们在六聚体形成、DNA依赖性ATP酶以及DNA解旋酶活性中的作用。缺失α结构域的突变体DnaBβγ蛋白形成了六聚体；而缺失γ结构域的突变体DnaBαβ只能形成二聚体。DnaBαβ的二聚化依赖于Mg(2+)。这些数据表明，DnaB解旋酶的寡聚化涉及至少两个不同的蛋白质 - 蛋白质相互作用位点；其中一个位点主要位于β结构域内（位点1），而另一个相互作用位点位于γ结构域内（位点2）。缺失α和γ结构域的147个氨基酸的多肽突变体DnaBβ表现出完全功能性的ATP酶活性。因此，该结构域构成了ATP酶活性的“中央催化结构域”。DnaBαβ的ATP酶活性在动力学上与DnaBβ相当，表明α结构域对ATP酶活性影响很小或没有影响。在这两种情况下，ATP酶活性均不依赖于DNA。DnaBβγ具有DNA依赖性ATP酶活性，其在动力学上与野生型DnaB蛋白（wtDnaB）的ATP酶活性相当，表明C末端γ结构域在增强β结构域的ATP酶活性以及DNA结合方面具有特定作用。缺失α结构域的突变体DnaBβγ没有任何解旋酶活性，这表明α结构域具有重要作用。本研究的主要发现是：（i）β结构域包含一个功能性ATP酶活性位点；（ii）γ结构域似乎是DNA结合结构域以及β结构域ATP酶活性的正调节因子；（iii）尽管α结构域对ATP酶、DNA结合活性或六聚体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旋DNA方面肯定起着关键作用；（iv）解旋酶活性需要所有三个结构域。
