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.