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

Abstract

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) KRASG13D, which suggests an exchange between two conformational states in solution. Methyl and 31P NMR spectra of active KRASG13D in solution confirm a two-state ensemble interconverting on the millisecond timescale, with a major Pγ 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 KRASG13D and KRASG13D-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 KRASG13D, 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.

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR

“…KRAS Q61L and G13D mutants also exhibit a predominant state 1 population. Chemical shifts of γ 1 and γ 2 peaks are not well resolved in the G13D spectrum because of their partial overlap and that could affect accurate population determination, as noted elsewhere ( 26 ). Reported binding affinities between the RAF1-RBD and WT and oncogenic mutants of KRAS-GppNHp follow the codon 12 rank order as (in decreasing order) G12V < G12D < G12C < WT, whereas Q61L and G13D proteins show higher affinities than G12V ( 27 ).…”

High Pressure Promotes Binding of the Allosteric Inhibitor Zn2+‐Cyclen in Crystals of Activated H‐Ras

“…This constitutes a proof of concept that the use of high pressure crystallography opens up new possibilities to study at atomic resolution drug binding to rare conformational states of proteins. Development of new drugs targeting specific pocket of different Ras isoforms, specially K‐Ras and mutants which are highly oncogenic, [56,57] would take advantage of high pressure crystallography to determine high resolution structures of high energy conformers, to visualize with great details ligand binding site in these excited states and enable more efficient ligand screening.…”

Producing recombinant proteins in Vibrio natriegens

“…A more striking example of differential protein folding is observed when comparing the chromatographic separation of the initial IMAC capture step for His6-MBP-tev-GG-Hs.KRAS4b(2-169) [20,29,30]. Figure 4A depicts the pertinent elution fractions (A 280 trace and SDS-PAGE/Coomassie staining analysis) and the subsequent TEV protease treatment of pooled fractions.…”