Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
J Biomol NMR. 2013 May;56(1):17-30. doi: 10.1007/s10858-013-9719-9. Epub 2013 Mar 3.
Many processes in the regulation of gene expression and signaling involve the formation of protein complexes involving multi-domain proteins. Individual domains that mediate protein-protein and protein-nucleic acid interactions are typically connected by flexible linkers, which contribute to conformational dynamics and enable the formation of complexes with distinct binding partners. Solution techniques are therefore required for structural analysis and to characterize potential conformational dynamics. Nuclear magnetic resonance spectroscopy (NMR) provides such information but often only sparse data are obtained with increasing molecular weight of the complexes. It is therefore beneficial to combine NMR data with additional structural restraints from complementary solution techniques. Small angle X-ray/neutron scattering (SAXS/SANS) data can be efficiently combined with NMR-derived information, either for validation or by providing additional restraints for structural analysis. Here, we show that the combination of SAXS and SANS data can help to refine structural models obtained from data-driven docking using HADDOCK based on sparse NMR data. The approach is demonstrated with the ternary protein-protein-RNA complex involving two RNA recognition motif (RRM) domains of Sex-lethal, the N-terminal cold shock domain of Upstream-to-N-Ras, and msl-2 mRNA. Based on chemical shift perturbations we have mapped protein-protein and protein-RNA interfaces and complemented this NMR-derived information with SAXS data, as well as SANS measurements on subunit-selectively deuterated samples of the ternary complex. Our results show that, while the use of SAXS data is beneficial, the additional combination with contrast variation in SANS data resolves remaining ambiguities and improves the docking based on chemical shift perturbations of the ternary protein-RNA complex.
许多基因表达和信号调控过程涉及涉及多结构域蛋白的蛋白质复合物的形成。介导蛋白质-蛋白质和蛋白质-核酸相互作用的各个结构域通常通过柔性接头连接，这有助于构象动力学，并能够与不同的结合伙伴形成复合物。因此，需要溶液技术进行结构分析和表征潜在的构象动力学。核磁共振波谱（NMR）提供了这种信息，但随着复合物分子量的增加，通常只能获得稀疏的数据。因此，将 NMR 数据与来自互补溶液技术的附加结构约束相结合是有益的。小角度 X 射线/中子散射（SAXS/SANS）数据可以与从 NMR 衍生的信息有效地结合，无论是用于验证还是为结构分析提供附加约束。在这里，我们表明，SAXS 和 SANS 数据的组合可以帮助从基于稀疏 NMR 数据的基于 HADDOCK 的数据驱动对接中获得的结构模型进行细化。该方法通过涉及 Sex-lethal 的两个 RNA 识别基序（RRM）结构域、Upstream-to-N-Ras 的 N 端冷休克结构域和 msl-2 mRNA 的三元蛋白质-蛋白质-RNA 复合物来证明。基于化学位移扰动，我们已经绘制了蛋白质-蛋白质和蛋白质-RNA 界面，并将此 NMR 衍生信息与 SAXS 数据以及三元复合物亚基选择性氘代样品的 SANS 测量结果进行了补充。我们的结果表明，尽管使用 SAXS 数据是有益的，但与 SANS 数据中的对比度变化相结合可以解决剩余的歧义，并提高基于三元蛋白质-RNA 复合物化学位移扰动的对接。
