Dynamic conformational equilibria in the active states of KRAS and NRAS

Abstract

The design of potent RAS inhibitors benefits from a molecular understanding of the dynamics in KRAS and NRAS and their oncogenic mutants. Here we characterize switch-1 dynamics in GTP-state KRAS and NRAS by ³¹P NMR, by ¹⁵N relaxation dispersion NMR, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and molecular dynamics simulations. In GMPPNP-bound KRAS and NRAS, we see the co-existence of two conformational states, corresponding to an "inactive" state-1 and an "active" state-2, as previously reported. The KRAS oncogenic mutations G12D, G12C and G12V only slightly affect this equilibrium towards the "inactive" state-1, with rank order wt < G12C < G12D < G12V. In contrast, the NRAS Q61R oncogenic mutation shifts the equilibrium fully towards the "active" state-2. Our molecular dynamics simulations explain this by the observation of a transient hydrogen bond between the Arg61 side chain and the Thr35 backbone carbonyl oxygen. NMR relaxation dispersion experiments with GTP-bound KRAS Q61R confirm a drastic decrease in the population of state-1, but still detect a small residual population (1.8%) of this conformer. HDX-MS indicates that higher populations of state-1 correspond to increased hydrogen-deuterium exchange rates in some regions and increased flexibility, whereas low state-1 populations are associated with KRAS rigidification. We elucidated the mechanism of action of a potent KRAS G12D inhibitor, MRTX1133. Binding of this inhibitor to the switch-2 pocket causes a complete shift of KRAS G12D towards the "inactive" conformation and prevents binding of effector RAS-binding domain (RBD) at physiological concentrations, by signaling through an allosteric network.

Fig. 1. (A) ³¹P NMR spectra of RAS isoforms and mutants as discussed in this paper and indicated in the Figure. (B) Structural differences between RAS state-1 and state-2 conformations, illustrated by the crystal structures of GMPPNP-bound HRAS in state-2 conformation (green, 5p21) and in state-1 conformation (white, 4efl). The green sphere represents a Mg²⁺ ion. The side chains of Tyr32 and Thr35 are shown. The transparent blue surface and cartoon illustrate the position of bound RBD and its steric clash with Tyr32 in state-1.

Fig. 2. Populations of state-1 (blue) and state-2 (peach) as determined by ³¹P NMR in (A) KRAS mutants, (B) RAS isoforms, (C) NRAS mutants, (D) KRAS G12D complexed with an inhibitor. All corresponding ³¹P spectra are shown in Fig. 1A. All RAS proteins were bound to GMPPNP and measurements were carried out at 7 °C. The asterisk (*) refers to previously published data on HRAS and MRAS.

Fig. 3. NMR data for ¹⁵N labeled WT (blue), T35S (green) and Q61R (red) KRAS bound to GTP. (A) Overlay of the glycine spectral region from HSQC spectra, 25 °C. The ‘×’ sign indicates the position of G12 and G13 for Q61R, indicating to a good approximation the positions of the cross-peaks for these residues in ‘pure’ state-2, while the ‘+’ sign indicates the expected cross-peak positions of the ‘pure’ state-1 using chemical shift differences calculated from fits of ¹H^N and ¹⁵N CPMG data for the WT protein. (B) ¹⁵N CPMG profiles for five selected residues. Circles indicate experimental data, while lines indicate fits. (C) Position of the two mutated residues (T35 and Q61), of the five ¹⁵N probes shown in panel (B), and of GMPPNP·Mg.

Fig. 4. Molecular dynamics (MD) simulations of KRAS wt (A), KRAS G12D (B), NRAS wt (C) and NRAS Q61R (D). The mobilities, seen as carbon-alpha RMSDs relative to the state-2 starting structures, of switch-1 and switch-2 are plotted on the horizontal and vertical axis, respectively. The typical RMSD of 1.3 Å after minimization of starting X-ray structures is shown as a cyan marker.

Fig. 5. Hydrogen–deuterium exchange mass spectrometry (HDX-MS). (A) Chiclet plots for NRAS T35S, Q61 and T35S/Q61R mutant proteins bound to GMPPNP, with NRAS wt GMPPNP taken as baseline. Numerical column values are time points given in minutes. White background indicates lacking data for the corresponding peptide comparisons due to mutation. Regions discussed in the text are labeled accordingly. (B) Coloring scale for chiclet plots: green = deprotection, blue = protection. (C) NRAS Q61R vs. wt comparison from (a) structurally mapped onto the crystal structure of KRAS Q61 GMPPNP, PBD 6XGU. Arg61 is shown in magenta carbons. (D) Chiclet plot for KRAS G12D GMPPNP vs. KRAS wt GMPPNP. All values used to make these graphs are found in the supplementary HDX data file (ESI†).

Fig. 6. Characterization of MRTX1133 binding to KRAS G12D. (A) ³¹P NMR spectra of KRAS G12D:GMPPNP (top) and KRAS G12D:GMPPNP in complex with MRTX1133 (bottom). (B) ¹H,¹³C-HMQC NMR spectra of GMPPNP-bound KRAS G12D (black), KRAS G12D + RBD (blue), KRAS G12D + MRTX1133 (green) and KRAS G12D + RBD + MRTX1133 (red). The concentrations were: KRAS G12D, 20 μM; MRTX1133, 40 μM; RBD, 60 μM. Note that RBD does not bind to the KRAS G12D/MRTX1133 complex at these concentrations. (C) Structural details on the allosteric relay of MRTX1133 binding from the switch-2 pocket to switch-1. Without MRTX1133 bound (PDB: 6QUU), the switch-2 loop folds such that Ala59's β-methyl points upwards. MRTX1133 binding leads to opening up of switch-2, formation of Asp12 and Gly60 ligand contacts, and rotation of Ala59 β-methyl such that switch-1 is disrupted with Thr35 rotating towards solvent, leading to an open conformation (PDB: 7T47). (D) HDX-MS chiclet plot for KRAS G12D:GMPPNP bound to compound 25 vs. KRAS G12D:GMPPNP alone. All values used to make these graphs are found in the supplementary HDX data file (ESI†). The coloring scheme is the same as in Fig. 4. Numerical column values are time points given in minutes. Compound 25 leads to strong protection across the protein. (E) HDX data structurally mapped onto the crystal structure of KRAS G12D GPPCP bound to MRTX1133 (PDB 7T47). D12 is shown in blue carbons.