Universidad de Buenos Aires, CONICET, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Junín 956, CABA 1113, Argentina.
Universidad de Buenos Aires, CONICET, Instituto de Cálculo, Intendente Guiraldes 2160, CABA 1428, Argentina.
Nucleic Acids Res. 2023 Nov 10;51(20):11213-11224. doi: 10.1093/nar/gkad808.
The presence of ATP is known to stimulate helicase activity of the Dengue Virus Non-structural protein 3 helicase (NS3h), and the presence of RNA stimulates NS3h ATPase activity, however this coupling is still mechanistically unclear. Here we use atomistic models and molecular dynamics simulations to evaluate the single-stranded RNA (ssRNA)-length dependence of the NS3h-ssRNA binding affinity and its modulation by bound ATP. Considering complexes with 7, 11, 16 and 26 nucleotides (nts), we observe that both the binding affinity and its modulation by bound ATP are augmented with increased ssRNA lengths. In models with at least 11 nts bound, the binding of ATP results in a shift from a tightly bound to a weakly bound state. We find that the weakly bound state persists during both the ADP-Pi- and ADP-bound stages of the catalytic cycle. We obtain the equilibrium association constants for NS3h binding to an ssRNA 10-mer in vitro, both in the absence and presence of ADP, which further support the alternation between tightly and weakly bound states during the catalytic cycle. The length of bound ssRNA is critical for understanding the NS3h-RNA interaction as well as how it is modulated during the catalytic cycle.
已知 ATP 的存在会刺激登革热病毒非结构蛋白 3 解旋酶（NS3h）的解旋酶活性，而 RNA 的存在会刺激 NS3h ATP 酶活性，但这种偶联在机制上仍不清楚。在这里，我们使用原子模型和分子动力学模拟来评估 NS3h-ssRNA 结合亲和力的单链 RNA（ssRNA）长度依赖性及其对结合 ATP 的调节。考虑到含有 7、11、16 和 26 个核苷酸（nts）的复合物，我们观察到结合亲和力及其对结合 ATP 的调节都随着 ssRNA 长度的增加而增加。在结合至少 11nt 的模型中，ATP 的结合导致从紧密结合状态转变为弱结合状态。我们发现，在催化循环的 ADP-Pi 和 ADP 结合阶段，弱结合状态都持续存在。我们获得了 NS3h 在体外与 ssRNA 10 -mer 结合的平衡缔合常数，无论是在没有 ADP 的情况下还是存在 ADP 的情况下，这进一步支持了催化循环中紧密结合态和弱结合态之间的交替。结合 ssRNA 的长度对于理解 NS3h-RNA 相互作用以及它在催化循环中如何被调节至关重要。
