Flexible-body motions of calmodulin and the farnesylated hypervariable region yield a high-affinity interaction enabling K-Ras4B membrane extraction

Abstract

In calmodulin (CaM)-rich environments, oncogenic KRAS plays a critical role in adenocarcinomas by promoting PI3K/Akt signaling. We previously proposed that at elevated calcium levels in cancer, CaM recruits PI3Kα to the membrane and extracts K-Ras4B from the membrane, organizing a K-Ras4B-CaM-PI3Kα ternary complex. CaM can thereby replace a missing receptor-tyrosine kinase signal to fully activate PI3Kα. Recent experimental data show that CaM selectively promotes K-Ras signaling but not of N-Ras or H-Ras. How CaM specifically targets K-Ras and how it extracts it from the membrane in KRAS-driven cancer is unclear. Obtaining detailed structural information for a CaM-K-Ras complex is still challenging. Here, using molecular dynamics simulations and fluorescence experiments, we observed that CaM preferentially binds unfolded K-Ras4B hypervariable regions (HVRs) and not α-helical HVRs. The interaction involved all three CaM domains including the central linker and both lobes. CaM specifically targeted the highly polybasic anchor region of the K-Ras4B HVR that stably wraps around CaM's acidic linker. The docking of the farnesyl group to the hydrophobic pockets located at both CaM lobes further enhanced CaM-HVR complex stability. Both CaM and K-Ras4B HVR are highly flexible molecules, suggesting that their interactions permit highly dynamic flexible-body motions. We, therefore, anticipate that the flexible-body interaction is required to extract K-Ras4B from the membrane, as conformational plasticity enables CaM to orient efficiently to the polybasic HVR anchor, which is partially diffused into the liquid-phase membrane. Our structural model of the CaM-K-Ras4B HVR association provides plausible clues to CaM's regulatory action in PI3Kα activation involving the ternary complex in cell proliferation signaling by oncogenic K-Ras.

Keywords: GTPase Kras (KRAS); KRAS-driven adenocarcinoma; PI3K; calmodulin (CaM); fluorescence; hypervariable region; molecular dynamics; nuclear magnetic resonance (NMR); post-translational modification (PTM); prenylation.

Figure 1.

Shown are crystal structures of CaM with a stretched central linker yielding an extended structure (PDB code 1CLL) (A) and a collapsed linker yielding a compact globular conformation (PDB code 1CDL) (B). CaM consists of two symmetric globular domains, N-lobe and C-lobe, connected by a flexible linker. C, each lobe of CaM contains a hydrophobic pocket. In the protein structures, hydrophobic, polar/glycine, positively charged, and negatively charged residues are colored white, green, blue, and red, respectively.

Figure 2.

Structures of typical binding partners for CaM. Shown is a crystal structure of CaM-binding α-chain domain (residues 1731–1749) from MYLK (A) and a modeled α-helix of K-Ras4B HVR (residues 167–185) with the post-translational modification (white stick) (B). The helical conformation of HVR was adopted from the crystal MYLK peptide structure. Initial configurations of compact globular CaMs (red) wrapping around the MYLK peptide (green) (C) and α-helical (D) and inverted (E) α-helical peptide of K-Ras4B HVR (blue). Blue spheres in CaM denote Ca²⁺, and white transparent surfaces in HVR represent the farnesyl group.

Figure 3.

Shown are relaxed structures of compact globular CaM embedding the α-helical binding partners in the aqueous environments for the MYLK peptide (green) (A) and α-helical (B) and inverted (C) α-helical peptide of K-Ras4B HVR (blue). Large conformational changes in CaM are apparent with the α-helical K-Ras4B HVRs by superimposition of the final (red) and initial (transparent white) conformations of CaM. D, time series of r.m.s.d. from the starting point for C_α atoms of CaM. Two dimensional dynamical cross-correlations maps of the residues motions across CaM with respect to the MYLK peptide (E) and α-helical (F) and inverted (G) α-helical peptide of K-Ras4B HVR.

Figure 4.

Shown are NMR CSPs of residues by the K-Ras4B HVR mapped onto the crystal structures of CaM with a stretched central linker (A) and a collapsed linker (B). In the structures, residues with high CSPs induced by non-farnesylated and farnesylated HVRs are marked by red and blue letters/surface, respectively. Initial configurations of CaM–K-Ras4B HVR complex are constructed using the crystal structures of CaM with a stretched central linker (configurations 1–4) (C) and a collapsed linker (configurations 5–8) (D). Unfolded HVRs are modeled in the complex, ensuring that the initial contacts of the HVR peptides on the CaM residues are determined by the NMR CSPs.

Figure 5.

Snapshots representing the relaxed CaM–K-Ras4B HVR complex (left panel of each configuration). Highlighted interfaces for the complex are shown (right panel of each configuration). In the highlighting, residues involved in the salt-bridge interactions are marked by yellow for CaM and cyan for K-Ras4B HVR.

Figure 6.

Highlights of the hydrophobic interactions of the farnesyl moiety (white stick) with the CaM residues (yellow sticks) at the hydrophobic pockets in the C-lobe of configuration 5 (upper panel) and the N-lobe of configuration 7 (lower panel) in a stereo representation.

Figure 7.

A, shown are the probabilities of salt-bridge formation for residues in CaM (left panel) and K-Ras4B HVR (right panel). B, probabilities of salt-bridge formation for the residue pair between CaM and K-Ras4B HVR. In the three-dimensional contour plot, those residues with high probability are selectively presented.

Figure 8.

Binding free energy for the CaM interaction with the K-Ras4B HVR. In the calculation, gas-phase contribution, ΔG_gas, the solvation energy contribution, ΔG_sol, and the entropic contribution, −TΔS, combines the average binding free energy, ΔG_b. In the box graphs, the blue and black horizontal lines denote the mean and median values, respectively.

Figure 9.

Examples of fluorescence spectrum (left panel) obtained after titrating for T27W (A), S82W (B), and Q137W (C) CaM mutants with different concentrations of the farnesylated K-Ras4B HVR peptide (beginning with 0.44–3 μm). The concentration of CaM was kept at 1.5 μm. Shown are dissociation constants for the N-terminal domain, linker region, and C-terminal domain obtained using nonlinear regression analysis (right panels). Fitting was done using a “one set of binding sites” model. a.u., arbitrary units.

Figure 10.

The difference in the cooperative-binding mode of CaM with different phospholipid types. A, extraction of the farnesylated K-Ras4B HVR peptide from DPPC phospholipids by CaM at low concentrations (< 0.2 μm) (left panel). Shown are control injections of unmodified HVR onto the DPPC nanodisc (right panel). B, at the same low concentration, CaM binds to the farnesylated HVR peptide and remains on the DOPC membrane (left panel). Shown is the control injection of unmodified HVR onto the DOPC nanodisc (right panel). C, at the same concentration, CaM bound farnesylated HVR and remained on the DPPS membrane (left panel). Shown is the control injection of unmodified HVR onto the DPPS nanodisc (right panel). D, at the same concentration, CaM bound farnesylated HVR and remained on the DOPS membrane (left panel). Shown is the control injection of unmodified HVR onto the DOPS nanodisc (right panel).

Figure 11.

Shown are time-series of snapshots of farnesylated HVR peptides of K-Ras4B interacting with CaM at the zwitterionic DPPC bilayer in the gel phase (A), the zwitterionic DOPC bilayer in the liquid phase (B), and the anionic DOPC:DOPS (mole ratio 4:1) bilayer in the liquid phase (C). In the HVR peptides, hydrophobic residues are shown in white, polar and Gly residues are shown in green, negatively charged residues are shown in red, and positively charged residues are in blue. The farnesyl group is shown as the thick yellow stick. In the CaM structure, the α-helix and β-sheet secondary structures are represented as red and yellow schematics, respectively. Shown is the time series of the deviation, Δz, from the averaged phosphate atom position at the upper bilayer leaflet as a function of time in a logarithm scale for the center of mass of the HVR peptide, selected carbon atoms in the farnesyl group, and the center of mass of CaM at the DPPC (D), the DOPC (E), and the DOPC:DOPS (mole ratio 4:1) (F) bilayers. The light gray area denotes the interior of the lipid bilayers. The location of the bilayer surface can be defined +5 Å from Δz = 0.

Figure 12.

The role of CaM in KRAS-driven adenocarcinoma. A schematic diagram illustrates K-Ras4B extraction from the membrane by CaM. With elevated Ca²⁺/CaM level, CaM selectively targets K-Ras4B in the active state. The active K-Ras4B removes autoinhibition, exposing its polybasic HVR. The K-Ras4B/CaM association recruits PI3Kα to the membrane and forms a K-Ras4B–CaM–PI3Kα ternary complex, substituting the missing receptor-tyrosine kinase signal.