The Importance of Mg²⁺‐Free State in Nucleotide Exchange of Oncogenic K‐Ras Mutants

Abstract

For efficient targeting of oncogenic K-Ras interaction sites, a mechanistic picture of the Ras-cycle is necessary. Herein, we used NMR relaxation techniques and molecular dynamics simulations to decipher the role of slow dynamics in wild-type and three oncogenic P-loop mutants of K-Ras. Our measurements reveal a dominant two-state conformational exchange on the ms timescale in both GDPand GTP-bound K-Ras. The identified low-populated higher energy state in GDP-loaded K-Ras has a conformation reminiscent of a nucleotide-bound/Mg 2 + -free state characterized by shortened β2/β3-strands and a partially released switch-I region preparing K-Ras for the interaction with the incoming nucleotide exchange factor and subsequent reactivation. By providing insight into mutation-specific differences in K-Ras structural dynamics, our systematic analysis improves our understanding of prolonged K-Ras signaling and may aid the development of allosteric inhibitors targeting nucleotide exchange in K-Ras.

Deciphering Conformational Changes of the GDP-Bound NRAS Induced by Mutations G13D, Q61R, and C118S through Gaussian Accelerated Molecular Dynamic Simulations

“…These results suggest that the HBIs of V29 and D30 in the SW I with GDP are highly unstable. The aforementioned interaction sites of GDP and NRAS agree well with the GDP binding spots identified in the work of Johnson et al [ 29 ].…”

“…As shown in Figure 1 A, SW I, SWII, and the P-loop encircle a binding pocket of GTP or GDP. The X-ray and NMR experiments by Pálfy et al revealed that the conformations of the switch domains from the RAS proteins are highly affected by G12 mutations [ 28 , 29 ]. Mutations lead to large conformational changes in the two switches, SW I and SW II, which yield a significant effect on the activity of NRAS [ 30 , 31 ].…”

Exploring the state- and allele-specific conformational landscapes of Ras: understanding their respective druggabilities

“…Ras oncoproteins can exist in different nucleotide-bound and mutant forms in cells, all of which are inherently flexible 7,29–33,54,55 populating a diverse set of conformations including many that possess druggable pockets unobserved in the ground structures. To date, studies that took advantage of the cryptic pockets 10,13–15,38,56 for targeting have significantly advanced the therapeutics development against Ras and proved covalent modification a feasible strategy for direct inhibition of Ras(G12C)·GDP.…”

“…26,27 Conformational exchange was also captured by relaxation-based NMR in the inactive GDP-bound state, which however occurs on a much faster timescale. 28 Previous molecular dynamics and enhanced sampling simulations 7,11,29–33 suggest that the cryptic pockets can commonly pre-exist in different nucleotide-bound and mutant forms. However, it remains unclear how the mutations and activation states alter the “invisible” vulnerable conformers of Ras leading to the differential druggabilities noted in recent drug development efforts.…”

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

“…Although several studies on RAS proteins by solution-state NMR have shown the presence of allostery connecting the effector and allosteric lobes using chemical shift perturbations 5 , 19 and relaxation dispersion experiments 20 – 22 , it is not clear how those observations can be translated to structural changes outside of the switch regions in KRAS in solution, which is the focus of our present NMR study. It had been shown that the dynamics of the HRAS isoform can be affected by using non-hydrolyzable GTP analogs 23 , and recent dynamic studies using GTP with HRAS 22 and with KRAS 24 showed that two different minor states of GTP-bound RAS can be produced under different experimental conditions. Interestingly, although HRAS and KRAS proteins have an identical primary sequence in the effector lobe, they display different structural features in the reported minor states, highlighting the complex dynamics of RAS proteins in solution.…”