Extending the Lifetime of Native GTP-Bound Ras for Site-Resolved NMR Measurements: Quantifying the Allosteric Dynamics.

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.

Characterization of the interaction interface and conformational dynamics of human TGIF1 homeodomain upon the binding of consensus DNA.

The TG interacting factor-1 homeodomain (TGIF1-HD) binds with the consensus DNA motif 5'-TGTCA-3' in gene promoters through its three-amino acid loop extension (TALE) type homeodomain, and then recruits co-regulators to regulate gene expression. Although the solution NMR structure of human TGIF1-HD has been reported previously, little is known about its DNA binding mechanism. NMR titrations have been extensively used to study mechanisms of ligand binding to target proteins; however, an intermediate exchange occurred predominantly between TGIF1-HD in the free and bound states when titrated with the consensus DNA, which resulted in poor-quality NMR spectra and precluded further exploration of its interaction interface and conformational dynamics.

Microsecond Timescale Dynamics of GDP-Bound Ras Underlies the Formation of Novel Inhibitor-Binding Pockets.

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.