Influence of Cholesterol on the Orientation of the Farnesylated GTP-Bound KRas-4B Binding with Anionic Model Membranes

Abstract

The Ras family of proteins is tethered to the inner leaflet of the cell membranes which plays an essential role in signal transduction pathways that promote cellular proliferation, survival, growth, and differentiation. KRas-4B, the most mutated Ras isoform in different cancers, has been under extensive study for more than two decades. Here we have focused our interest on the influence of cholesterol on the orientations that KRas-4B adopts with respect to the plane of the anionic model membranes. How cholesterol in the bilayer might modulate preferences for specific orientation states is far from clear. Herein, after analyzing data from in total 4000 ns-long molecular dynamics (MD) simulations for four KRas-4B systems, properties such as the area per lipid and thickness of the membrane as well as selected radial distribution functions, penetration of different moieties of KRas-4B, and internal conformational fluctuations of flexible moieties in KRas-4B have been calculated. It has been shown that high cholesterol content in the plasma membrane (PM) favors one orientation state (OS1), exposing the effector-binding loop for signal transduction in the cell from the atomic level. We confirm that high cholesterol in the PM helps KRas-4B mutant stay in its constitutively active state, which suggests that high cholesterol intake can increase mortality and may promote cancer progression for cancer patients. We propose that during the treatment of KRas-4B-related cancers, reducing the cholesterol level in the PM and sustaining cancer progression by controlling the plasma cholesterol intake might be taken into account in anti-cancer therapies.

Predicting the Conformational Variability of Oncogenic GTP-bound G12D Mutated KRas-4B Proteins at Cell Membranes

“…The free-energy profiles of conformational orientations of the mutated and methylated protein computed by WTM have given us quantitative information on the energy barriers that must be surmounted by KRas-4B to change its orientation. We considered as CV of the method two angles previously defined 31 , able to clearly distinguish variations of the orientation of the protein. These two angles (Φ, Θ) are able to describe two classes of angular reorientations: one related to changes of one single coordinate and a more complex one corresponding to joint changes of the two CVs.…”

“…The energy barrier of such TS between A and B will indicate the amount of free-energy required for KRas-4B-FMe to exchange its conformation between the two principal orientations, as it was described in Ref. 31 . The two stable states A and B are represented in Fig.…”

“…2. They were considered to describe the conformational changes along free-energy paths, as recently described in a study devoted to KRas-4B-Far proteins anchoring to the anionic model membranes with different cholesterol concentrations 31 .…”

“…In the present work, given the shorter durability and strenght of the interactions of FAR and the cell membrane, we have taken a different approach to analyze the free-energy landscape of KRas-4B-FMe. Following studies showing the significant influence of orientations of Ras proteins on membranes related to their function in cell and considering that cell membranes are platforms for cellular signal transduction 82,[86][87][88][89] we reported in a recent study 31 two preferential orientations of oncogenic and wild-type KRas-4B-Far proteins able to describe the reorientations of the protein while anchored at the membrane. Henceforth, we considered the two particular orientations, Θ and Φ defined above as the CV of the WTM method, we have run 975 ns WTM simulations and obtained the corresponding free-energy hypersurface.…”

“…Further, free-energy barriers along with the corresponding free-energy landscape (FEL) for KRas-4B-Far binding with the anionic model membrane were described. The main orientational angles of KRas-4B-Far when bound to the cell membrane in a fully quantative fashion 31 have been recently reported. In the present work, our aim has been to investigate the alternative farnesylated and methylated KRas-4B species (KRas-4B-FMe) and its structural, orientational and energetic characteristics.…”