PIP2 Influences the Conformational Dynamics of Membrane bound KRAS4b.

Abstract

KRAS4b is a small GTPase involved in cellular signaling through receptor tyrosine kinases. The activation of KRAS4b only occurs after recruitment of the regulatory proteins to the plasma membrane, thus making the role of the phospholipid bilayer an integral part of the signaling mechanism. Phospholipids, primarily with anionic head groups, interact with both the membrane anchoring hypervariable (HVR) region and the G-(catalytic) domain, and influence the orientation of KRAS4b on the membrane surface, potentially play-ing a key role in the regulation of activation. Although there has been significant research focused on the role of the anionic phosphatidyl serine, less effort has been made on the role of the important signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Using instrumentation to measure the fluorescence anisotro-py decay of site specifically labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) Nanodiscs over a wide frequency range, we measure the binding of KRAS4b to Nanodiscs containing either 30% phosphati-dylserine (PS) or 10% L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) by measuring the rotational corre-lation time of the Nanodisc-KRAS4b complex. We find that KRAS4b binds significantly tighter to Nanodiscs containing PIP2, but that at any level of binding saturation of KRAS4b, both 30% PS and 10% PIP2 con-taining Nanodiscs display similar rotational correlation times. This shows that the overall hydrodynamic ra-dii of the KRAS4b-Nanodisc complexes are similar regardless of the incorporated anionic lipid. Atomic force microscopy (AFM) is used to visualize KRAS4b when bound to individual Nanodiscs. Clean images are observed with the PIP2 doped Nanodiscs, but significantly blurred images are obtained when the anionic lipid is PS. This suggests that the KRAS4b is not only more tightly bound overall with PIP2 as the anionic lipid, but is also less mobile on the bilayer surface. Microsecond molecular dynamics simulations of KRAS4b on PS and PIP2 containing membranes show that the dynamics of the G-domain at the bilayer sur-face are significantly altered in the presence of PIP2, due to the formation of long lived salt bridges with basic residues on the G-domain. The orientation and dynamics of KRAS4b on the membrane is critical to understanding the mechanisms of oncoprotein signaling and our results with the GDP-bound form show subtle differences from that published for GTP-KRAS4b.