Defining the ATPase center of bacteriophage T4 DNA packaging machine: Requirement for a catalytic glutamate residue in the large terminase protein gp17
Defining the ATPase center of bacteriophage T4 DNA packaging machine: Requirement for a catalytic glutamate residue in the large terminase protein gp17
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DOI：
10.1016/s0022-2836(03)00636-3
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发表时间：
2003-08-01
影响因子：
5.6
通讯作者：
Rao, VB
中科院分区：
文献类型：
--
作者：
Goetzinger, KR;Rao, VB
二十面体噬菌体中的双链DNA包装由ATP酶偶联的包装机驱动，所述包装机由门蛋白和在空病毒衣壳的独特门顶点处组装的两个非结构包装/末端酶蛋白构成。最近的研究表明，噬菌体T4大末端酶蛋白gp 17的N-末端ATP酶位点是DNA包装所必需的。很可能这是DNA易位ATP酶，其驱动DNA定向易位到病毒衣壳中。因此，定义这个ATP酶中心对于理解ATP驱动的病毒DNA易位机制至关重要。使用组合诱变和生物化学方法，我们已经定义了催化ATP水解所需的羧酸残基。尽管最初的催化羧酸盐假说表明在步行者A（SRQLGKT（161-167））和步行者B（MIYID 251 -255）基序之间存在催化谷氨酸盐，但四个候选谷氨酸残基E198、E208、E220和E227中没有一个是功能所需的。然而，紧邻推定的步行者B天冬氨酸残基（D255）的E256残基表现出与催化羧酸功能一致的表型模式。没有一个氨基酸取代，包括高度保守的D和Q，是耐受的。生化分析表明，纯化的E256 V，D和Q突变体gp 17表现出完全丧失的gp 16刺激的ATP酶活性和体外DNA包装活性，而它们的ATP结合和DNA切割功能保持完整。这些数据表明，E256突变体被困在ATP结合的构象，不能催化ATP水解-转导循环，动力DNA易位。因此，这项研究首次鉴定和表征了参与病毒DNA包装机能量转导机制的催化谷氨酸残基。(C)2003 Elsevier Ltd.保留所有权利。
Double-stranded DNA packaging in icosahedral bacteriophages is driven by an ATPase-coupled packaging machine constituted by the portal protein and two non-structural packaging/terminase proteins assembled at the unique portal vertex of the empty viral capsid. Recent studies show that the N-terminal ATPase site of bacteriophage T4 large terminase protein gp17 is critically required for DNA packaging. It is likely that this is the DNA translocating ATPase that powers directional translocation of DNA into the viral capsid. Defining this ATPase center is therefore fundamentally important to understand the mechanism of ATP-driven DNA translocation in viruses. Using combinatorial mutagenesis and biochemical approaches, we have defined the catalytic carboxylate residue that is required for ATP hydrolysis. Although the original catalytic carboxylate hypothesis suggested the presence of a catalytic glutamate between the Walker A (SRQLGKT(161-167)) and Walker B (MIYID251-255) motifs, none of the four candidate glutamic acid residues, E198, E208, E220 and E227, is required for function. However, the E256 residue that is immediately adjacent to the putative Walker B aspartic acid residue (D255) exhibited a phenotypic pattern that is consistent with the catalytic carboxylate function. None of the amino acid substitutions, including the highly conservative D and Q, was tolerated. Biochemical analyses showed that the purified E256V, D, and Q mutant gp17s exhibited a complete loss of gp16-stimulated ATPase activity and in vitro DNA packaging activity, whereas their ATP binding and DNA cleavage functions remained intact. The data suggest that the E256 mutants are trapped in an ATP-bound conformation and are unable to catalyze the ATP hydrolysis-transduction cycle that powers DNA translocation. Thus, this study for the first time identified and characterized a catalytic glutamate residue that is involved in the energy transduction mechanism of a viral DNA packaging machine. (C) 2003 Elsevier Ltd. All rights reserved.