Campus Chemical Instrument Center, The Ohio State University, Columbus, OH, USA.
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA.
Nat Struct Mol Biol. 2023 Oct;30(10):1446-1455. doi: 10.1038/s41594-023-01070-z. Epub 2023 Aug 28.
Despite the prominent role of the K-Ras protein in many different types of human cancer, major gaps in atomic-level information severely limit our understanding of its functions in health and disease. Here, we report the quantitative backbone structural dynamics of K-Ras by solution nuclear magnetic resonance spectroscopy of the active state of wild-type K-Ras bound to guanosine triphosphate (GTP) nucleotide and two of its oncogenic P-loop mutants, G12D and G12C, using a new nanoparticle-assisted spin relaxation method, relaxation dispersion and chemical exchange saturation transfer experiments covering the entire range of timescales from picoseconds to milliseconds. Our combined experiments allow detection and analysis of the functionally critical Switch I and Switch II regions, which have previously remained largely unobservable by X-ray crystallography and nuclear magnetic resonance spectroscopy. Our data reveal cooperative transitions of K-Ras·GTP to a highly dynamic excited state that closely resembles the partially disordered K-Ras·GDP state. These results advance our understanding of differential GTPase activities and signaling properties of the wild type versus mutants and may thus guide new strategies for the development of therapeutics.
尽管 K-Ras 蛋白在许多不同类型的人类癌症中都起着重要作用，但在原子水平信息方面仍存在重大差距，这严重限制了我们对其在健康和疾病中的功能的理解。在这里，我们通过使用一种新的纳米颗粒辅助的自旋弛豫方法，对与鸟苷三磷酸 (GTP) 核苷酸结合的野生型 K-Ras 以及其两种致癌 P 环突变体 G12D 和 G12C 的活性状态进行了溶液核磁共振波谱学研究，报道了 K-Ras 的定量骨架结构动力学。我们的综合实验允许检测和分析功能关键的 Switch I 和 Switch II 区域，这些区域以前在 X 射线晶体学和核磁共振波谱学中基本上是不可观察的。我们的数据揭示了 K-Ras·GTP 向高度动态激发态的协同转变，该状态与部分无序的 K-Ras·GDP 状态非常相似。这些结果增进了我们对野生型与突变体的 GTP 酶活性和信号转导特性的理解，从而可能为开发治疗方法提供新的策略。
