Reduced dynamic complexity allows structure elucidation of an excited state of KRASG13D

Structural analysis reveals an intermediate (excited) state of GMPPNP-bound KRAS(G13D) which includes the formation of a new pocket in the allosteric lobe that could be exploited for developing new therapeutics against KRAS(G13D)-driven cancers. Localized dynamics of RAS, including regions distal to the nucleotide-binding site, is of high interest for elucidating the mechanisms by which RAS proteins interact with effectors and regulators and for designing inhibitors. Among several oncogenic mutants, methyl relaxation dispersion experiments reveal highly synchronized conformational dynamics in the active (GMPPNP-bound) KRAS(G13D), which suggests an exchange between two conformational states in solution. Methyl and P-31 NMR spectra of active KRAS(G13D) in solution confirm a two-state ensemble interconverting on the millisecond timescale, with a major P-gamma atom peak corresponding to the dominant State 1 conformation and a secondary peak indicating an intermediate state different from the known State 2 conformation recognized by RAS effectors. High-resolution crystal structures of active KRAS(G13D) and KRAS(G13D)-RAF1 RBD complex provide snapshots of the State 1 and 2 conformations, respectively. We use residual dipolar couplings to solve and cross-validate the structure of the intermediate state of active KRAS(G13D), showing a conformation distinct from those of States 1 and 2 outside the known flexible switch regions. The dynamic coupling between the conformational exchange in the effector lobe and the breathing motion in the allosteric lobe is further validated by a secondary mutation in the allosteric lobe, which affects the conformational population equilibrium.