Ding Jienyu, Deme Justin C, Stagno Jason R, Yu Ping, Lea Susan M, Wang Yun-Xing
Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Molecular Basis of Disease Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Nucleic Acids Res. 2023 Oct 13;51(18):9952-9960. doi: 10.1093/nar/gkad651.
RNA conformational heterogeneity often hampers its high-resolution structure determination, especially for large and flexible RNAs devoid of stabilizing proteins or ligands. The adenosylcobalamin riboswitch exhibits heterogeneous conformations under 1 mM Mg2+ concentration and ligand binding reduces conformational flexibility. Among all conformers, we determined one apo (5.3 Å) and four holo cryo-electron microscopy structures (overall 3.0-3.5 Å, binding pocket 2.9-3.2 Å). The holo dimers exhibit global motions of helical twisting and bending around the dimer interface. A backbone comparison of the apo and holo states reveals a large structural difference in the P6 extension position. The central strand of the binding pocket, junction 6/3, changes from an 'S'- to a 'U'-shaped conformation to accommodate ligand. Furthermore, the binding pocket can partially form under 1 mM Mg2+ and fully form under 10 mM Mg2+ within the bound-like structure in the absence of ligand. Our results not only demonstrate the stabilizing ligand-induced conformational changes in and around the binding pocket but may also provide further insight into the role of the P6 extension in ligand binding and selectivity.
RNA构象异质性常常阻碍其高分辨率结构的测定，尤其是对于缺乏稳定蛋白或配体的大型柔性RNA。腺苷钴胺素核糖开关在1 mM Mg2+浓度下呈现异质构象，而配体结合会降低构象灵活性。在所有构象体中，我们确定了一个无配体（5.3 Å）和四个有配体的冷冻电子显微镜结构（整体分辨率为3.0 - 3.5 Å，结合口袋分辨率为2.9 - 3.2 Å）。有配体的二聚体在二聚体界面周围呈现螺旋扭曲和弯曲的整体运动。无配体和有配体状态的主链比较显示，P6延伸位置存在较大的结构差异。结合口袋的中央链，即6/3连接处，从“S”形构象转变为“U”形构象以容纳配体。此外，在没有配体的情况下，结合口袋在1 mM Mg2+浓度下可部分形成，在10 mM Mg2+浓度下可在类似结合的结构中完全形成。我们的结果不仅证明了稳定配体诱导结合口袋及其周围的构象变化，还可能为P6延伸在配体结合和选择性中的作用提供进一步的见解。
