NMR experiments probe mechanism and binding site.

<p>(A) ³¹P-NMR spectrum of non-prenylated GMPPNP-loaded KRAS^G12V shows two environments for the γ-phosphate (top) with a shift to a single inactive population with 1 mM compound <b>2</b> (middle) and a single active population with 0.5 mM of the CRAF-RBD (bottom); α and β phosphate have single environments and the KRAS^G12V is present at 5 mg/ml. (B) ¹⁵N,¹H-HSQC spectrum for non-prenylated GDP-KRAS^G12V in absence (black) or presence (blue) of 400 μM compound <b>2</b> (protein concentration is 0.5 mg/ml). The residues with major chemical shift changes are indicated with circles. (C) Residues engaged in nOe’s between non-prenylated GDP-KRAS^G12V and compound <b>2</b> shown in blue stick model with labels using the crystal structure for compound <b>6</b> (PDB-ID 4EPY) as reference; compound <b>6</b> in orange, GDP and RAS protein in green, switch-I in magenta and switch-II in yellow.