Long-lasting Salt Bridges Provide the Anchoring Mechanism of Oncogenic Kirsten Rat Sarcoma Proteins at Cell Membranes

Abstract

RAS proteins work as GDP-GTP binary switches and regulate cytoplasmic signalling networks that are able to control several cellular processes, playing an essential role in signal transduction pathways involved in cell growth, differentiation and survival so that overacting RAS signalling can lead to cancer. One of the hardest challenges to face is the design of mutation-selective therapeutic strategies. In this work, a G12D mutated farnesylated GTP bound Kirsten RAt Sarcoma (KRAS) protein has been simulated at the interface of a DOPC/DOPS/cholesterol model anionic cell membrane.A specific long-lasting salt bridge connection between farnesyl and the hypervariable region of the protein has been identified as the main mechanism responsible of the binding of oncogenic farnesylated KRAS-4B to the cell membrane. Free-energy landscapes allowed us to characterise local and global minima of KRAS-4B binding to the cell membrane revealing the main pathways between anchored and released states.

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

“…S7 and S8 of Ref. 19 ) which help to stabilize KRas-4B-Far binding with the interface of the membrane, regardless of its farnesyl group anchoring into the membrane or not. In the present case, we have observed that under the circumstances of phosphorylation at Ser-181, the O f me of farnesyl group forms typical HB with the phenoxyl hydrogen atom belonging to Tyr137 of CD (see Fig.…”

“…Since oncogenic Ras is defective in GAP-mediated GTP hydrolysis it results in an ac-residues 1-166, in a sequence which contains a catalytic lobe (the effector-binding domain, residues 1-86) and an allosteric lobe (residues . HVR preferentially binds the membrane in the liquid crystal phase, by inserting its farnesyl moiety into the phospholipid bilayer 18,19 . Ras proteins are distinguished by their unique HVR post-translational modifications (PTM).…”

“…In a recent work a detailed study of the demethylated KRas-4B-Far was reported 19 . It was observed that strong, long-lasting salt-bridges between the FAR and HVR moieities are able to stabilize the binding of the oncogenic KRas-4B-Far to the PM for long periods of time, of the order of microseconds.…”

“…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. We have employed all-atom molecular dynamics (MD) and the FEL have been obtained by and well-tempered metadynamics (WTM) simulations 19 , in order to acquire precise information on the orientation of the full-length KRas-4B and its preferential conformations when binding with the anionic bilayers. The proposed angular variables will be employed as collective variables (i.e.…”

“…2, we can observe that the mutant KRas-4B-FMe spends most of the time in the active state centered around (1.0 rad, 1.6 rad), exposing the effector-binding domain for further signal transduction. Obviously, in the last 500 ns trajectories of MD simulations, the mutated KRas-4B-FMe is trapped in the active state because of the strong interactions between HVR and the interface of the bilayer, 19 and seldom does KRas-4B-FMe explore other conformations blocked by high energy barriers. We continued to explore the corresponding free-energy landscape by conducting WTM simulations.…”