Yang Teng-Chieh, Ortiz David, Nosaka Lyn'Al, Lander Gabriel C, Catalano Carlos Enrique
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington.
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California.
Biophys J. 2015 Oct 20;109(8):1663-75. doi: 10.1016/j.bpj.2015.08.037.
Viral terminase enzymes serve as genome packaging motors in many complex double-stranded DNA viruses. The functional motors are multiprotein complexes that translocate viral DNA into a capsid shell, powered by a packaging ATPase, and are among the most powerful molecular motors in nature. Given their essential role in virus development, the structure and function of these biological motors is of considerable interest. Bacteriophage λ-terminase, which serves as a prototypical genome packaging motor, is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. This protomer assembles into a functional higher-order complex that excises a unit length genome from a concatemeric DNA precursor (genome maturation) and concomitantly translocates the duplex into a preformed procapsid shell (genome packaging). While the enzymology of λ-terminase has been well described, the nature of the catalytically competent nucleoprotein intermediates, and the mechanism describing their assembly and activation, is less clear. Here we utilize analytical ultracentrifugation to determine the thermodynamic parameters describing motor assembly and define a minimal thermodynamic linkage model that describes the effects of salt on protomer assembly into a tetrameric complex. Negative stain electron microscopy images reveal a symmetric ring-like complex with a compact stem and four extended arms that exhibit a range of conformational states. Finally, kinetic studies demonstrate that assembly of the ring tetramer is directly linked to activation of the packaging ATPase activity of the motor, thus providing a direct link between structure and function. The implications of these results with respect to the assembly and activation of the functional packaging motor during a productive viral infection are discussed.
病毒末端酶在许多复杂的双链DNA病毒中充当基因组包装马达。功能性马达是多蛋白复合物，由包装ATP酶提供动力，将病毒DNA转运到衣壳中，是自然界中最强大的分子马达之一。鉴于它们在病毒发育中的重要作用，这些生物马达的结构和功能备受关注。噬菌体λ末端酶作为典型的基因组包装马达，由一个与两个DNA识别亚基紧密结合的大催化亚基组成。该原体组装成一个功能性的高阶复合物，从串联DNA前体中切除单位长度的基因组（基因组成熟），并同时将双链转运到预先形成的原衣壳中（基因组包装）。虽然λ末端酶的酶学已得到充分描述，但具有催化活性的核蛋白中间体的性质以及描述其组装和激活的机制尚不清楚。在这里，我们利用分析超速离心来确定描述马达组装的热力学参数，并定义一个最小热力学连锁模型，该模型描述了盐对原体组装成四聚体复合物的影响。负染电子显微镜图像显示了一个对称的环状复合物，有一个紧凑的茎和四个伸出的臂，呈现出一系列构象状态。最后，动力学研究表明，环状四聚体的组装与马达包装ATP酶活性的激活直接相关，从而在结构和功能之间建立了直接联系。本文讨论了这些结果对生产性病毒感染期间功能性包装马达组装和激活的意义。
