Chemistry Program, Science Division, New York University, Abu Dhabi, United Arab Emirates.
Department of Chemistry, New York University, USA.
Nucleic Acids Res. 2023 Nov 10;51(20):11332-11344. doi: 10.1093/nar/gkad809.
SARS-CoV-2 depends on -1 programmed ribosomal frameshifting (-1 PRF) to express proteins essential for its replication. The RNA pseudoknot stimulating -1 PRF is thus an attractive drug target. However, the structural models of this pseudoknot obtained from cryo-EM and crystallography differ in some important features, leaving the pseudoknot structure unclear. We measured the solution structure of the pseudoknot using small-angle X-ray scattering (SAXS). The measured profile did not agree with profiles computed from the previously solved structures. Beginning with each of these solved structures, we used the SAXS data to direct all atom molecular dynamics (MD) simulations to improve the agreement in profiles. In all cases, this refinement resulted in a bent conformation that more closely resembled the cryo-EM structures than the crystal structure. Applying the same approach to a point mutant abolishing -1 PRF revealed a notably more bent structure with reoriented helices. This work clarifies the dynamic structures of the SARS-CoV-2 pseudoknot in solution.
SARS-CoV-2 依赖于 -1 核糖体移码（-1 PRF）来表达其复制所必需的蛋白质。因此，刺激 -1 PRF 的 RNA 假结是一个有吸引力的药物靶点。然而，从 cryo-EM 和晶体学获得的这个假结的结构模型在一些重要特征上存在差异，使得假结结构不清楚。我们使用小角 X 射线散射（SAXS）测量了假结的溶液结构。测量的轮廓与以前解决的结构计算的轮廓不相符。从每个已解决的结构开始，我们使用 SAXS 数据来指导全原子分子动力学（MD）模拟，以提高轮廓的一致性。在所有情况下，这种改进导致了一种弯曲构象，与 cryo-EM 结构更相似，而不是晶体结构。将相同的方法应用于消除 -1 PRF 的点突变体揭示了一种明显更弯曲的结构，其螺旋重新定向。这项工作阐明了 SARS-CoV-2 假结在溶液中的动态结构。
