Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC 27708.
Department of Diagnostic and Biological Sciences, University of Minnesota, Minneapolis, MN 55455.
Proc Natl Acad Sci U S A. 2021 Apr 27;118(17). doi: 10.1073/pnas.2024928118.
Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors.
许多病毒利用环状包装 ATP 酶在复制过程中将双链 DNA 转运到前衣壳中。这种 ATP 酶的机械化学循环的关键步骤是 ATP 结合，这导致马达中的一个亚基紧紧抓住 DNA。在这里，我们探讨了 ATP 结合与 DNA 结合偶联的潜在分子机制，并表明在病毒包装 ATP 酶中，AAA+ 酶中的谷氨酸开关残基是这种偶联的核心。我们使用通过分子动力学模拟计算得出的自由能景观，确定了 ATP 和 ADP 结合状态下 ATP 酶活性位点的稳定构象状态。我们的结果表明，催化谷氨酸残基在 ATP 水解时从活性构象转变为非活性构象，而被指定为谷氨酸开关的残基对于调节这种转变是必需的。此外，我们通过互信息分析确定了由谷氨酸开关介导的分子内信号通路，该通路负责将 ATP 结合与 DNA 结合构象转变偶联。我们通过结构和功能实验测量证实了这些预测。具体来说，我们表明 ADP 结合的 P74-26 包装 ATP 酶的晶体结构与模拟预测的结构偶联一致，并且我们进一步表明，破坏预测的信号通路确实使 ATP 酶活性与 φ29 DNA 包装马达中的 DNA 易位活性解耦。我们的工作因此建立了一种信号通路，将病毒 DNA 包装马达中的化学和机械事件偶联起来。
