Probe-based molecular dynamics (pMD) simulation is a useful approach to determine druggable binding sites. The premise of pMD is similar to multi-solvent crystallography or NMR fragment-based screening. Applying pMD in the presence of membrane poses a challenge because the probe molecules can partition into the bilayer affecting its structure. We developed an approach, pMD-membrane, where we modify the force field parameters to alleviate this effect. This involves reducing the pairwise non-bonded interaction between selected probe and lipid atoms. We applied pMD-membrane to identify allosteric binding sites in a well-known cancer target, Ras. Ras is a lipid-modified GTPase and is involved in a plethora of signaling pathways. Membrane binding is essential for Ras biological function. Mutations in Ras are associated with a variety of cancers including pancreatic and colorectal cancer. Of the three major human Ras isoforms H-, N- and K-Ras, cancers associated with mutant K-Ras are the most lethal. Although there is strong evidence for the existence of different orientations of H-Ras with respect to membrane plane, the data on K-Ras was not conclusive. Therefore, first, we performed ~8μs all-atom MD simulations of full-length oncogenic (G12D) K-Ras bound to a heterogeneous membrane. MD-derived populations revealed that the K-Ras catalytic domain interacts directly with the membrane with two predominant and distinct modes of interaction. Of the two modes only one was found capable of effector binding. Next, we applied pMD-membrane on the two orientations of membrane-bound K-Ras and different K-Ras mutants, G13D and G12D. The results suggest differential dynamics of the allosteric pockets significantly affect accessibility to probes. Using these and other examples, we will discuss how pMD-membrane can be used to track isoform-dependent or mutation-induced differences in the ligand binding potential of pockets on the surface of Ras proteins.

pMD-membrane: A tool to determine allosteric binding pockets in membrane-bound biomolecules

251st National Meeting (2016)

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