Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7
Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7
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DOI：
10.1074/jbc.m110.156067
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发表时间：
2010-10-08
影响因子：
4.8
通讯作者：
Richardson, Charles C.
中科院分区：
文献类型：
--
作者：
Lee, Seung-Joo;Richardson, Charles C.
DNA解旋酶在单链DNA上的移位和双链DNA的解旋由核苷三磷酸（NTP）的水解提供动力。虽然大多数解旋酶在这些过程中使用ATP，但由噬菌体T7的基因4编码的DNA解旋酶最有效地使用dTTP。为了确定NTP的结构要求，我们使用各种NTP及其类似物确定了T7解旋酶的DNA解旋效率。胸腺嘧啶的5-甲基对于DNA的有效解旋至关重要，尽管3 '-核糖基羟基的存在部分克服了这一要求。通过用氨基酸随机取代晶体结构表明与核苷酸相互作用的几个氨基酸残基（Thr-320、Arg-504、Tyr-535和Leu-542）来检查蛋白质的NTP结合口袋。虽然位置320和542需要适当大小的脂肪族残基，但在位置535处需要芳香族侧链以稳定NTP以进行有效解旋。残基504的碱性侧链对于与dTTP的胸腺嘧啶碱基的4-羰基相互作用是必不可少的。用一个小的脂肪族残基取代这个残基允许容纳其他NTP，导致优先使用dATP和使用dCTP，一种通常不使用的核苷酸。这项研究的结果表明，NTP必须通过蛋白质的NTP结合位点内的特异性相互作用来稳定，以实现有效的水解。这些相互作用决定了NTP特异性。
The translocation of DNA helicases on single-stranded DNA and the unwinding of double-stranded DNA are fueled by the hydrolysis of nucleoside triphosphates (NTP). Although most helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To identify the structural requirements of the NTP, we determined the efficiency of DNA unwinding by T7 helicase using a variety of NTPs and their analogs. The 5-methyl group of thymine was critical for the efficient unwinding of DNA, although the presence of a 3'-ribosyl hydroxyl group partially overcame this requirement. The NTP-binding pocket of the protein was examined by randomly substituting amino acids for several amino acid residues (Thr-320, Arg-504, Tyr-535, and Leu-542) that the crystal structure suggests interact with the nucleotide. Although positions 320 and 542 required aliphatic residues of the appropriate size, an aromatic side chain was necessary at position 535 to stabilize NTP for efficient unwinding. A basic side chain of residue 504 was essential to interact with the 4-carbonyl of the thymine base of dTTP. Replacement of this residue with a small aliphatic residue allowed the accommodation of other NTPs, resulting in the preferential use of dATP and the use of dCTP, a nucleotide not normally used. Results from this study suggest that the NTP must be stabilized by specific interactions within the NTP-binding site of the protein to achieve efficient hydrolysis. These interactions dictate NTP specificity.