Dargis Rokas, Pajak Joshua, Ariyawansa Pavan, Morais Marc C, Jardine Paul J, Arya Gaurav
Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC 27708.
Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605.
Proc Natl Acad Sci U S A. 2025 Jul 8;122(27):e2504780122. doi: 10.1073/pnas.2504780122. Epub 2025 Jul 3.
Certain viruses such as tailed bacteriophages and herpes simplex virus package double-stranded DNA into empty procapsids via powerful, ring-shaped molecular motors. High-resolution structures and force measurements on the DNA packaging motor of bacteriophage Φ29 revealed that its five ATPase subunits coordinate ATP hydrolysis with each other to maintain the proper cyclic sequence of DNA translocation steps about the ring. Here, we explore how the Φ29 motor regulates translocation by timing key events, namely ATP binding/hydrolysis and DNA gripping, through trans-subunit interactions. We used subunit dimers bound to DNA as our model system, a minimal system that still captures the conformation and trans-subunit interactions of the full pentameric motor complex. Molecular dynamics simulations of all-ATP and mixed ATP-ADP dimers revealed that the nucleotide occupancy of one subunit strongly affects the ability to hydrolyze ATP in the adjacent subunit by altering the free energy landscape of its catalytic glutamate approaching the gamma phosphate of ATP. Specifically, one ATP-bound subunit donates residues in trans that sterically block the neighboring subunit's catalytic glutamate. This steric hindrance is resolved when the first subunit hydrolyzes ATP and is ADP bound. This obstructive mechanism is supported by functional mutagenesis and appears to be conserved across several Φ29 relatives. Mutual information analysis of our simulations revealed intersubunit signaling pathways, via the trans-acting obstructive residues, that allow for sensing and communication between the binding pockets of adjacent subunits. This work reveals how the sequential order of DNA translocation events among subunits is preserved through trans-subunit interactions and pathways.
某些病毒，如尾部噬菌体和单纯疱疹病毒，通过强大的环形分子马达将双链DNA包装到空的原衣壳中。对噬菌体Φ29的DNA包装马达进行的高分辨率结构和力测量表明，其五个ATP酶亚基相互协调ATP水解，以维持DNA围绕环进行转运步骤的正确循环序列。在这里，我们探讨了Φ29马达如何通过亚基间相互作用对关键事件（即ATP结合/水解和DNA抓握）进行计时来调节转运。我们使用与DNA结合的亚基二聚体作为我们的模型系统，这是一个最小的系统，仍然能够捕捉完整的五聚体马达复合物的构象和亚基间相互作用。对全ATP和混合ATP-ADP二聚体的分子动力学模拟表明，一个亚基的核苷酸占据情况会通过改变其催化谷氨酸接近ATPγ磷酸的自由能态势，强烈影响相邻亚基水解ATP的能力。具体来说，一个结合ATP的亚基会在反式方向上提供残基，在空间上阻碍相邻亚基的催化谷氨酸。当第一个亚基水解ATP并结合ADP时，这种空间位阻就会消除。功能诱变支持了这种阻碍机制，并且这种机制似乎在几个Φ29的亲属中是保守的。我们模拟的互信息分析揭示了通过反式作用阻碍残基的亚基间信号通路，该通路允许相邻亚基的结合口袋之间进行传感和通信。这项工作揭示了亚基间的相互作用和信号通路是如何保持亚基间DNA转运事件的顺序的。
