Multisubunit viral ATPases that couple ATP-hydrolysis to genome translocation

描述(申请人提供)：病毒基因组的包装是生物学中的一个基本过程。在许多DNA病毒中，这一反应是由一个大的(约1.5MDa)基因组包装马达推动的，该马达是由一个终止酶全酶组装成一个十二聚体门户蛋白形成的。这种大分子复合体的功能就像一个化学马达，它可以分解ATP，以高达每秒2000个碱基的速度将病毒基因组的副本转移到预先形成的衣壳内。除了是自然界中最快、最强大的引擎外，基因组包装马达也是一台耐人寻味的分子机器，在结构和催化机制方面都不是很好。在这项资助中，我们将使用结晶学和生化技术的组合来表征噬菌体P22基因组包装马达的结构。在我实验室最近确定的P22门户蛋白结构的基础上，我们的工作将集中在P22终止酶的化学上，这是一种在发动机中起作用的ATPase。P22终止酶与门户蛋白复合体的结构特征将提供一个结构框架，以破译在包装马达内，终止酶如何将ATP水解酶与病毒基因组的易位偶联。此外，由于终止酶在疱疹病毒中高度保守，这项拨款中建议的工作也将提供一个逻辑框架，开始对致病人类病毒包装马达的结构特征进行表征。我们工作的具体目标是：1.确定组装在门户蛋白顶端的P22终止酶全酶的结构；确定在基因组包装过程中，小末端酶亚基如何刺激大末端酶ATPase活性。 与公共卫生相关：病毒基因组转移马达是多亚单位三磷酸腺苷酶，它燃烧三磷酸腺苷来驱动病毒基因组在预先形成的衣壳内的包装。在这项资助中，我们建议研究噬菌体P22基因组易位马达的结构和活性。我们的工作将确定自然界中最小的发动机之一是如何将化学能量与基因组易位结合在一起的。

DESCRIPTION (provided by applicant): Packaging of viral genomes is a fundamental process in biology. In many DNA viruses this reaction is powered by a large (M.W. ~1.5MDa) genome-packaging motor, which is formed by a terminase holoenzyme assembled to a dodecameric portal protein. This macromolecular complex functions like a chemical motor, which hydrolyzes ATP to translocate a copy of the viral genome inside a preformed capsid, at rates as high as 2000 bases per second. In addition to being the fastest and most powerful engine in nature, the genome-packaging motor is also an intriguing molecular machine, which is poorly characterized both in structure and catalytic mechanisms. In this grant, we will use a combination of crystallographic and biochemical techniques to characterize the structure of the bacteriophage P22 genome-packaging motor. Building upon the structure of P22 portal protein, which was recently determined in my laboratory, our work will focus on the chemistry of P22 terminase that is a functional ATPase in the motor. The structural characterization of P22 terminase in complex with portal protein will provide a structural framework to decipher how, within the packaging motor, terminase couples ATP hydrolysis to translocation of viral genomes. In addition, since terminases are highly conserved in herpesviruses, the work proposed in this grant will also provide a logical framework to begin structural characterization of packaging motors in pathogenic human viruses. Specific aims of our work are: 1.) to determine the structure of the P22 terminase holoenzyme assembled at the portal protein vertex; 2.) to define how small terminase subunit stimulates large terminase ATPase activity during genome-packaging. PUBLIC HEALTH RELEVANCE: Viral genome-translocating motors are multisubunit ATPases that burn ATP to power packaging of viral genomes inside a preformed capsid. In this grant, we propose to study the structure and activity of the bacteriophage P22 genome-translocating motor. Our work will determine how one of smallest motors in nature couples chemical energy to genome translocation.