Conformational and Dynamical Effects of Tyr32 Phosphorylation in K-Ras: Molecular Dynamics Simulation and Markov State Models Analysis

Abstract

Phosphorylation of tyrosine 32 in K-Ras has been shown to influence function by disrupting the GTPase cycle. To shed light on the underlying mechanism and atomic basis of this process, we carried out a comparative investigation of the oncogenic G12D K-Ras mutant and its phosphorylated variant (pTyr32) using all-atom molecular dynamics simulations and Markov state models. We show that, despite sharing a number of common features, G12D and pTyr32-G12D K-Ras exhibit some distinct conformational states and fluctuations. In addition to notable differences in conformation and dynamics of residues surrounding the GTP binding site, nonlocal changes were observed at a number of loops. Switch I is more flexible in pTyr32-G12D K-Ras while switch II is more flexible in G12D K-Ras. We also used time-lagged independent component analysis and k-means clustering to identify five metastable states for each system. We utilized transition path theory to calculate the transition probabilities for each state to build a Markov state model for each system. These models and other close inspections suggest that the phosphorylation of Tyr32 strongly affects protein dynamics and the active site conformation, especially with regards to the canonical switch conformations and dynamics.

Active and Inactive Cdc42 Differ in Their Insert Region Conformational Dynamics

“…We also show that the mutants affect the binding site exposure to the solvent and the interactions with other amino acids, Tyr32 and Thr35, near the binding site. This may indicate that the mutants play a similar role in GTP hydrolysis as in K-Ras4A and K-Ras4B and other Ras proteins ( 41 , 58 , 59 , 60 , 61 ).…”

Q61 mutant-mediated dynamics changes of the GTP-KRAS complex probed by Gaussian accelerated molecular dynamics and free energy landscapes

“…The previous works suggested that mutations signicantly affect the conformational alterations of the switch-I and switch-II in KRAS and change binding activity of KRAS to its regulators and effectors. [25][26][27][28][29][30][31][32][33] From residue substitutions at different sites, G12, G13 and Q61 mutations respectively account for 89%, 9% and 1% of the observed mutants of KRAS. 34 In addition, the effect of mutations at the other sites on the GTP-and GDP-KRAS binding were also investigated by different work groups.…”

The Q61H mutation decouples KRAS from upstream regulation and renders cancer cells resistant to SHP2 inhibitors

“…phosphorylation alters the conformational dynamics of the KRAS protein 51 . Furthermore, the phosphorylation of WT-KRAS caused a substantial allosteric effects on the dynamics of the bound RAF1-RBD.…”

“…The details of each system are presented in Supplementary Table 3 . The MD simulations were conducted following previously published approaches with some modification 51 , 79 . At the preparation phase, each system was placed in a solvent-filled cube of TIP3P water molecules with a solvent padding size of 20 Å in each dimension.…”