Excited state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants

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 K-Ras function in health and disease. Here, we report the quantitative backbone structural dynamics of K-Ras by solution NMR spectroscopy of the active state of wild-type K-Ras·GTP and two of its oncogenic P-loop mutants, G12D and G12C, using a novel nanoparticle-assisted spin relaxation method, relaxation dispersion and chemical exchange saturation transfer experiments covering the entire range of timescales from picosecond to milliseconds. Our combined experiments allow the detection and analysis of the functionally critical Switch I and Switch II regions that have previously remained largely unobservable by X-ray crystallography and NMR 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 o..., NMR relaxation dispersion experiments were acquired on an 850 MHz Bruker magnet equipped with a 5 mm TCI cryoprobe and a 600 MHz Bruker magnet equipped with a 5 mm TXI cryoprobe at 298 K. Amide 15N CPMG experiments were acquired at both magnetic fields using either the CW-CPMG or STCW-CPMG pulse sequences. The constant relaxation time was set to 40 ms and the CPMG pulsing frequency, νCPMG, was varied from 25 Hz to 2 kHz on the 850 MHz instrument, and from 25 Hz to 1 kHz on the 600 MHz instrument. Amide 1HN CPMG experiments were acquired using the sequence of Yuwen and Kay. A constant relaxation time of 16 ms was used and νCPMG was varied from 62.5 Hz to 4 kHz at the 850 MHz instrument only. Amide 15N CEST experiments were performed for all samples on the 850 MHz instrument using a CEST mixing time of 150 ms and B1 field strengths of ~40 Hz. Additional amide HSQC/HMQC experiments were acquired at both 600 and 850 MHz to aid in the sign determination of small 15N CPMG-derived chemical shi..., All relaxation dispersion data are provided as text files that can be used by the ChemEx software.
A table of relaxation rates and S2 values is provided for NASR results.

尽管K-Ras蛋白（K-Ras protein）在多种人类癌症中发挥着关键作用，但原子级信息的重大缺口严重限制了我们对K-Ras在健康与疾病中功能的认知。本研究通过溶液核磁共振波谱法（solution NMR spectroscopy），结合新型纳米颗粒辅助自旋弛豫方法、弛豫弥散（relaxation dispersion）与化学交换饱和转移实验（chemical exchange saturation transfer experiments），覆盖皮秒至毫秒的全时间尺度，解析了野生型K-Ras·GTP活性态及其两种致癌P环突变体（oncogenic P-loop mutants）G12D、G12C的主链结构动力学定量特征。我们的联合实验得以检测并分析功能关键的Switch I与Switch II区域（Switch I and Switch II regions）——此前X射线晶体学（X-ray crystallography）与核磁共振波谱法基本无法观测这些区域。数据显示，K-Ras·GTP会协同转变为高度动态的激发态，该状态与部分无序的K-Ras·GDP构象高度相似。本研究结果推动了相关领域发展[原文此处存在截断]。
本研究在配备5 mm TCI低温探头的850 MHz Bruker磁体，以及配备5 mm TXI低温探头的600 MHz Bruker磁体上，于298 K下开展了NMR弛豫弥散实验。酰胺15N CPMG实验在两个磁场下开展，采用CW-CPMG或STCW-CPMG脉冲序列。恒定弛豫时间设为40 ms，850 MHz仪器的CPMG脉冲频率νCPMG范围为25 Hz至2 kHz，600 MHz仪器为25 Hz至1 kHz。酰胺1HN CPMG实验采用Yuwen与Kay的序列，仅在850 MHz仪器上开展，恒定弛豫时间为16 ms，νCPMG范围为62.5 Hz至4 kHz。所有样品的酰胺15N CEST实验均在850 MHz仪器上开展，CEST混合时间为150 ms，B1场强约为40 Hz。此外在600和850 MHz下采集了额外的酰胺HSQC/HMQC实验，以辅助确定由15N CPMG得到的微小化学位移的符号[原文此处存在截断]。
所有弛豫弥散数据均以文本文件形式提供，可通过ChemEx软件进行分析。同时提供了NASR结果的弛豫速率与S2值表格。