The DEAD-box RNA helicase (DDX) plays a central role in many aspects of RNA metabolism by remodeling the defined structure of RNA molecules. While a number of structural studies have revealed the atomistic details of the interaction between DDX and RNA ligands, the molecular mechanism of how this molecule unwinds a structured RNA into an unstructured single-stranded RNA (ssRNA) has largely remained elusive. This is due to challenges in structurally characterizing the unwinding intermediate state and the lack of thermodynamic details underlying this process. In this study, we use solution nuclear magnetic resonance (NMR) spectroscopy to characterize the interaction of human DDX3X, a member of the DDX family, with various RNA ligands. Our results show that the inherent binding affinity of DDX3X for ssRNA is significantly higher than that for structured RNA elements. This preferential binding, accompanied by the formation of a domain-closed conformation in complex with ssRNA, effectively stabilizes the denatured ssRNA state and thus underlies the unwinding activity of DDX3X. Our results provide a thermodynamic and structural basis for the DDX function, whereby DDX can recognize and remodel a distinct set of structured RNAs to participate in a wide range of physiological processes.

DEAD-box RNA 解旋酶 (DDX) 通过重塑 RNA 分子的特定结构，在 RNA 代谢的许多方面发挥着核心作用。虽然许多结构研究揭示了 DDX 和 RNA 配体之间相互作用的原子细节，但该分子如何将结构化 RNA 解旋成非结构化单链 RNA (ssRNA) 的分子机制在很大程度上仍然难以捉摸。这是由于在结构表征解旋中间态方面面临的挑战以及缺乏该过程背后的热力学细节。在本研究中，我们使用溶液核磁共振 (NMR) 光谱来表征 DDX 家族成员人类 DDX3X 与各种 RNA 配体的相互作用。我们的结果表明，DDX3X 对 ssRNA 的固有结合亲和力明显高于对结构化 RNA 元件的结合亲和力。这种优先结合，伴随着与 ssRNA 复合物的结构域闭合构象的形成，有效地稳定了变性的 ssRNA 状态，从而成为 DDX3X 解旋活性的基础。我们的结果为 DDX 功能提供了热力学和结构基础，DDX 可以识别和重塑一组不同的结构化 RNA，以参与广泛的生理过程。