KRas4b-Calmodulin Interaction with Membrane Surfaces: Role of Headgroup, Acyl Chain, and Electrostatics

Abstract

KRas4b is a small plasma membrane-bound G-protein that regulates signal transduction pathways. The interaction of KRas4b with the plasma membrane is governed by both its basic C-terminus, which is farnesylated and methylated, and the lipid composition of the membrane itself. The signaling activity of KRas4b is intricately related to its interaction with various binding partners at the plasma membrane, underlining the critical role played by the lipid environment. The calcium-binding protein calmodulin binds farnesylated KRas4b and plays an important role in the dynamic spatial cycle of KRas4b trafficking in the cell. We utilize Biolayer Interferometry to assay the role of lipid headgroup, chain length, and electrostatics in the dissociation kinetics of fully post-translationally modified KRas4b from Nanodisc bilayers with defined lipid compositions. Our results suggest that calmodulin promotes the dissociation of KRas4b from an anionic membrane, with a comparatively slower displacement of KRas4b from PIP2 relative to PS containing bilayers. In addition to this headgroup dependence, KRas4b dissociation appears to be slower from Nanodiscs wherein the lipid composition contains mismatched, unsaturated acyl chains as compared to lipids with a matched acyl chain length. These findings contribute to understanding the role of the lipid composition in the binding of KRas4b and release from lipid bilayers, showing that the overall charge of the bilayer, the identity of the headgroups present, and the length and saturation of the acyl chains play key roles in KRas4b release from the membrane, potentially providing insights in targeting Ras-membrane interactions for therapeutic interventions.

Figure 1.

Measured KRas4b off-rates in the presence of varying concentrations of PDEδ and calmodulin. The varying anionic lipid compositions of 30 PS, 50 PS, and 10 PIP2 represent the anionic lipid composition of Nanodiscs (mol %) with DMPC as the background. Error bars represent the standard deviation of the experiments from two independent experimental replicates. (A) Off-rates as a function of PDEδ concentration as well as in the buffer. Each bar represents the off-rates (s⁻¹) of KRas4b-FME from the membrane surface, measured as a function of the PDEδ concentration (μM) for varying percentages of DMPS and PIP2 in the Nanodisc surface. (B) Dissociation rates of KRas4b-FME from Nanodiscs are measured in the presence of calmodulin without Ca²⁺ in the buffer. Each bar represents the off-rates (s⁻¹) of KRas4b-FME from the membrane surface, measured as a function of CaM concentration (μM) without Ca²⁺ for varying mole percentages of DMPS and PIP2 on the Nanodisc surface. (C) KRas4b-FME off-rates as a function of the CaM concentration in the presence of Ca²⁺. Each bar represents the off-rates (s⁻¹) of KRas4b-FME from the membrane surface, measured as a function of CaM concentration (μM) with Ca²⁺ in solution for varying percentages of DMPS and PIP2 in the Nanodisc surface.

Figure 2.

Representative plot of KRas4b-FME dissociation rates as a function of CaM concentration. Both panels represent the off-rate (s⁻¹) of KRas4b-FME from the membrane surface, measured as a function of CaM concentration (μM) in the presence of 1 mM Ca²⁺ for two different lipid headgroups: DOPS (A) and PIP2 (B) on a DMPC background. (A) Labels 10 DOPS, 30 DOPS, and 50 DOPS of DOPS represent the anionic composition of Nanodiscs (mol %) with the DMPC background. (B) Labels 2.5 PIP2, 5 PIP2, and 10 PIP2 represent the PIP2 anionic lipid composition (mol %) with the DMPC background. Results from two replicates are shown, with the line indicating a simple least-squares fit.

Figure 3.

Second-order KRas4b-FME off-rates (μM⁻¹ s⁻¹) from Nanodiscs of varied lipid compositions as a function of Nanodisc charge. (A) DMPS/DMPC (red), DOPS/DOPC (green), POPS/POPC (purple), and DPPS/DPPC (orange) represent second-order KRas4b-FME off-rates as a function of charge of bilayer on varied lipid compositions with matching acyl chains. (B) Comparing KRas4b-FME off-rates from POPS/POPC (purple) and DOPS/DOPC (green) to DOPS/DMPC (black) as a function of the charge of the bilayer. (C) Comparing KRas4b-FME off-rates from DOPS/DMPC (black) to PIP2/DMPC (blue) and PIP2/DOPC (teal) as a function of the total electrostatic charge of the bilayer. Results from two replicates are shown, with the line indicating a weighted least-squares fit due to the high variance in the 10% PS data because of the fast off-rates that approach the instrumentation limit.

Figure 4.

Schematic illustration of KRas4b dissociating from the lipid bilayer (Nanodisc) containing anionic lipids. KRas4b is removed from the lipid bilayer via CaM/Ca²⁺-mediated extraction at elevated levels of free Ca²⁺ or spontaneous dissociation. CaM becomes active upon binding to free calcium in solution. Upon depletion of free Ca²⁺, the globular domains of CaM are not exposed and cannot bind to free KRas4b. Anionic lipids may constrain KRas4b at the bilayer surface. KRas4b can bind to PDEδ in solution after dissociation from the lipid bilayer.