Characterization of native GTP‐bound Ras is important for an appreciation of its cellular signaling and for the design of inhibitors, which however has been depressed by its intrinsic instability. Herein, an effective approach for extending the lifetime of Ras⋅GTP samples by exploiting the active role of Son of Sevenless (Sos) is demonstrated that sustains the activated state of Ras. This approach, combined with a postprocessing method that suppresses residual Ras⋅GDP signals, is applied to the site‐resolved NMR measurement of the allosteric dynamics of Ras⋅GTP. The observed network of concerted motions well covers the recently identified allosteric inhibitor‐binding pockets, but the motions are more confined than those of Ras⋅GppNHp, advocating the use of native GTP for development of allosteric inhibitors. The Sos‐based approach is anticipated to generally facilitate experiments on active Ras when native GTP is preferred.

The recent discovery of inhibitory compounds binding to distinct pockets on GDP-bound Ras has renewed the view on the druggability of this crucial cancer driver. However, the origin of these pockets, which are not readily formed in the crystal structure in the absence of the compounds, is yet unclear. Herein, we explored the intrinsic flexibility of Ras⋅GDP on microsecond to millisecond timescales using relaxation-based NMR experiments, and identified substantial slow dynamics with τ of 34 μs at 5 °C, which maps to the regions showing a high level of correlation with those displaying conformational differences between the inhibitor-bound and free states. These findings, which have been demonstrated in both wild-type Ras and the oncogenic mutant (G12V), support the mechanism of extended conformational selection for Ras-inhibitor interactions where the small molecules redistribute the protein conformational ensemble favoring the final bound states.

Characterization of native GTP-bound Ras is important for an appreciation of its cellular signaling and for the design of inhibitors,which however has been depressed by its intrinsic instability.H erein, an effective approach for extending the lifetime of Ras·GTP samples by exploiting the active role of Son of Sevenless (Sos) is demonstrated that sustains the activated state of Ras.T his approach,c ombined with ap ostprocessing method that suppresses residual Ras·GDP signals,i sa pplied to the site-resolved NMR measurement of the allosteric dynamics of Ras·GTP.The observed network of concerted motions well covers the recently identified allosteric inhibitor-binding pockets,b ut the motions are more confined than those of Ras·GppNHp,advocating the use of native GTP for development of allosteric inhibitors.T he Sos-based approach is anticipated to generally facilitate experiments on active Ras when native GTP is preferred.

The recent discovery of inhibitory compounds binding to distinct pockets on GDP-bound Ras has renewed the view on the druggability of this crucial cancer driver. However,t he origin of these pockets,w hicha re not readily formed in the crystal structure in the absence of the compounds,i sy et unclear.H erein, we explored the intrinsic flexibility of Ras·GDP on microsecond to millisecond timescales using relaxation-based NMR experiments,and identified substantial slow dynamics with t ex of 34 msat58 8C, whichmaps to the regions showing ah igh level of correlation with those displaying conformational differences between the inhibitorbound and free states.T hese findings,w hich have been demonstrated in both wild-type Ras and the oncogenic mutant (G12V), support the mechanism of extended conformational selection for Ras-inhibitor interactions where the small molecules redistribute the protein conformational ensemble favoring the final bound states.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.