Goetzinger Katherine R, Rao Venigalla B
Department of Biology, The Catholic University of America, 103 McCort Ward Hall, 620 Michigan Ave, NE Washington, DC 20064, USA.
J Mol Biol. 2003 Aug 1;331(1):139-54. doi: 10.1016/s0022-2836(03)00636-3.
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 (MIYID(251-255)) 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.
二十面体噬菌体中的双链DNA包装由一个与ATP酶偶联的包装机器驱动，该机器由门户蛋白和两个非结构包装/末端酶蛋白组成，它们组装在空病毒衣壳的独特门户顶点处。最近的研究表明，噬菌体T4大末端酶蛋白gp17的N端ATP酶位点对于DNA包装至关重要。很可能这就是驱动DNA定向转运到病毒衣壳中的DNA转运ATP酶。因此，确定这个ATP酶中心对于理解病毒中ATP驱动的DNA转运机制至关重要。通过组合诱变和生化方法，我们确定了ATP水解所需的催化羧酸盐残基。尽管最初的催化羧酸盐假说是在沃克A（SRQLGKT(161 - 167)）和沃克B（MIYID(251 - 255)）基序之间存在一个催化谷氨酸，但四个候选谷氨酸残基E198、E208、E220和E227中没有一个是功能所必需的。然而，紧邻假定的沃克B天冬氨酸残基（D255）的E256残基表现出与催化羧酸盐功能一致的表型模式。包括高度保守的D和Q在内的任何氨基酸取代都不能被容忍。生化分析表明，纯化的E256V、D和Q突变型gp17s表现出gp16刺激的ATP酶活性和体外DNA包装活性完全丧失，而它们的ATP结合和DNA切割功能仍然完整。数据表明，E256突变体被困在ATP结合构象中，无法催化为DNA转运提供动力的ATP水解 - 转导循环。因此，这项研究首次鉴定并表征了一个参与病毒DNA包装机器能量转导机制的催化谷氨酸残基。
