DEAD-box helicases use ATP to unwind short double-stranded RNA (dsRNA). The helicase core consists of two discrete domains, termed RecA_N and RecA_C. The nucleotide binding site is harbored in RecA_N, while both RecA_N and RecA_C are involved in RNA recognition and ATP hydrolysis. In the absence of nucleotides or RNA, RecA_N and RecA_C do not interact (“open” form of the enzyme). In the presence of both RNA and ATP the two domains come together (“closed” form), building the composite RNA binding site and stimulating ATP hydrolysis. Because of the different roles and thermodynamic properties of the ADP-bound and ATP-bound states in the catalytic cycle, the conformations of DEAD-box helicases in complex with ATP and ADP are assumed to be different. However, the available crystal structures do not recapitulate these supposed differences and show identical conformations of DEAD-box helicases independent of the identity of the bound nucleotide. Here, we use NMR to demonstrate that the conformations of the ATP- and ADP-bound forms of the DEAD-box helicase Vasa are indeed different, contrary to the results from x-ray crystallography. These differences do not relate to the populations of the open and closed forms, but are intrinsic to the RecA_N domain. NMR chemical shift analysis reveals the regions of RecA_N where the average conformations of Vasa-ADP and Vasa-ATP are most different and indicates that these differences may contribute to modulating the affinity of the two nucleotide-bound complexes for RNA substrates.

中文翻译：

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DEAD-box 解旋酶 Vasa 核苷酸结合状态的长程构象变化


DEAD-box 解旋酶使用 ATP 解旋短双链 RNA （dsRNA）。解旋酶核心由两个离散结构域组成，称为 RecA_N 和 RecA_C。核苷酸结合位点隐藏在 RecA_N 中，而 RecA_N 和 RecA_C 都参与 RNA 识别和 ATP 水解。在没有核苷酸或 RNA 的情况下，RecA_N 和 RecA_C 不会相互作用（酶的“开放”形式）。在 RNA 和 ATP 存在的情况下，两个结构域聚集在一起（“封闭”形式），构建复合 RNA 结合位点并刺激 ATP 水解。由于 ADP 结合态和 ATP 结合态在催化循环中的作用和热力学性质不同，因此假设与 ATP 和 ADP 复合的 DEAD-box 解旋酶的构象是不同的。然而，可用的晶体结构并不能概括这些假定的差异，并显示出 DEAD-box 解旋酶的相同构象，与结合核苷酸的身份无关。在这里，我们使用 NMR 来证明 DEAD-box 解旋酶 Vasa 的 ATP 和 ADP 结合形式的构象确实不同，这与 X 射线晶体学的结果相反。这些差异与开放和封闭形式的总体无关，而是 RecA_N 域所固有的。NMR 化学位移分析揭示了 Vasa-ADP 和 Vasa-ATP 的平均构象差异最大的RecA_N区域，并表明这些差异可能有助于调节两个核苷酸结合复合物对 RNA 底物的亲和力。