Multisubunit viral ATPases that couple ATP-hydrolysis to genome translocation

描述（由申请人提供）：病毒基因组的包装是生物学的一个基本过程。在许多DNA病毒中，这种反应是由一个大的（m.w ~1.5MDa）基因组包装马达驱动的，它是由一个组装成十二聚体门脉蛋白的端酶全酶形成的。这种大分子复合物的功能就像一个化学马达，它水解ATP，以每秒高达2000个碱基的速度将病毒基因组的一个拷贝转移到预先形成的衣壳内。除了是自然界中最快、最强大的发动机外，基因组包装马达也是一种有趣的分子机器，其结构和催化机制的特征都很差。在这项资助中，我们将使用晶体学和生化技术的结合来表征噬菌体P22基因组包装马达的结构。在我的实验室最近确定的P22门静脉蛋白结构的基础上，我们的工作将集中在P22端酶的化学性质上，P22端酶是马达中的一种功能性atp酶。P22末端酶与门脉蛋白复合物的结构特征将提供一个结构框架，以解释在包装马达内，末端酶如何将ATP水解偶联到病毒基因组的易位。此外，由于疱疹病毒的末端高度保守，本基金提出的工作也将提供一个逻辑框架，开始对致病性人类病毒的包装马达进行结构表征。我们工作的具体目的是：1)确定在门静脉蛋白顶点组装的P22末端酶全酶的结构；2.)定义在基因组包装过程中，小端酶亚基如何刺激大端酶atp酶活性。

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.