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Abstract
Ras proteins are membrane-anchored GTPases that regulate key cellular signaling networks. It has been recently shown that different anionic lipid types can affect the properties of Ras in terms of dimerization/clustering on the cell membrane. To understand the effects of anionic lipids on key spatiotemporal properties of dimeric K-Ras4B, we perform all-atom molecular dynamics simulations of the dimer K-Ras4B in the presence and absence of Raf[RBD/CRD] effectors on two model anionic lipid membranes: one containing 78% mol DOPC, 20% mol DOPS, and 2% mol PIP2 and another one with enhanced concentration of anionic lipids containing 50% mol DOPC, 40% mol DOPS, and 10% mol PIP2. Analysis of our results unveils the orientational space of dimeric K-Ras4B and shows that the stability of the dimer is enhanced on the membrane containing a high concentration of anionic lipids in the absence of Raf effectors. This enhanced stability is also observed in the presence of Raf[RBD/CRD] effectors although it is not influenced by the concentration of anionic lipids in the membrane, but rather on the ability of Raf[CRD] to anchor to the membrane. We generate dominant K-Ras4B conformations by Markov state modeling and yield the population of states according to the K-Ras4B orientation on the membrane. For the membrane containing anionic lipids, we observe correlations between the diffusion of K-Ras4B and PIP2 and anchoring of anionic lipids to the Raf[CRD] domain. We conclude that the presence of effectors with the Raf[CRD] domain anchoring on the membrane as well as the membrane composition both influence the conformational stability of the K-Ras4B dimer, enabling the preservation of crucial interface interactions.
Supplementary materials
Title
Supporting Information
Description
Methods; lists of simulations, diffusion coefficients, and MSM parameters (Tables S1−S3); Ras dimerization conformation, structures, Chapman−Kolmogorov tests, timescales vs lag time and energy surfaces, RMSD time series and dimer simulations, percentage or orientations, membrane interactions, and membrane contacts vs number of residues, RMSF of the dimer and amino acids, distance vs time for salt bridges, secondary structure vs simulation time, frequency of the existence of a secondary structure element, mean square displacement plot and log(MSD(t))/log(t) plots, and correlation of lateral diffusion (Figures S1−S43).
Actions
Title
PDB files of cluster representatives and MSM states.
Description
Atomic coordinates of cluster representatives and Markov State Model macrostates (see Supporting Information for the procedures used to generate the structures).
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