A "Tug of War" Maintains a Dynamic Protein-Membrane Complex: Molecular Dynamics Simulations of C-Raf RBD-CRD Bound to K-Ras4B at an Anionic Membrane

Abstract

Association of Raf kinase with activated Ras triggers downstream signaling cascades toward regulating transcription in the cells' nucleus. Dysregulation of Ras-Raf signaling stimulates cancers. We investigate the C-Raf RBD and CRD regions when bound to oncogenic K-Ras4B at the membrane. All-atom molecular dynamics simulations suggest that the membrane plays an integral role in regulating the configurational ensemble of the complex. Remarkably, the complex samples a few states dynamically, reflecting a competition between C-Raf CRD- and K-Ras4B- membrane interactions. This competition arises because the interaction between the RBD and K-Ras is strong while the linker between the RBD and CRD is short. Such a mechanism maintains a modest binding for the overall complex at the membrane and is expected to facilitate fast signaling processes. Competition of protein-membrane contacts is likely a common mechanism for other multiprotein complexes, if not multidomain proteins at membranes.

Figure 1

C-Raf^RBD-CRD–K-Ras4B complex at a mixed membrane consisting of 80% POPC and 20% POPS. The K-Ras4B protein consists of the globular, catalytic domain (CD, res. 1–166) and the largely unstructured hypervariable region, HVR (res. 167–185). C-Raf comprises the Ras Binding Domain, RBD and Cysteine Rich Domain, CRD, connected by a short linker (res. 132–137). Proteins shown as mainchain cartoon; K-Ras (blue); RBD (orange); CRD (red); small molecules/ions as space filling: farnesyl group (gray); GTP (purple); Zn and Mg (tan); shown as lines: linker region (blue); HVR (green), membrane (blue); water (light cyan).

Figure 2

Configurations of C-Raf^RBD-CRD (C-Raf) in solution. (a) Starting structure. (b) Clustering of configurations with an RMSD cutoff distance of 5 Å, (c) representative configurations superimposed on RBD (pointing away from observer). Clusters #1–4 are shown (for other clusters, see Figure S1a). Surface electrostatic potential of domains (in configuration as seen in a), rotated by +90° around z.

Figure 3

Membrane binding dynamics of the C-Raf–K-Ras4B complex. (a–b) Time evolution of the distance of the center of K-Ras4B CD2 (residues 87–166) or the center of C-Raf CRD to the membrane center. Snapshots taken at various time points from (c) simulation #1 and (d) simulation #3. Color scheme is the same as in Figure 1 except POPC is in cyan, and POPS is in purple.

Figure 4

Interfaces of the K-Ras4B–membrane and C-Raf–membrane interactions. (a) Frequency of K-Ras4B–membrane contacts (residues within 5 Å of membrane surface); position of helices 3–5 is indicated. Inset images show two major orientations of complex relative to the membrane. (b) Frequency of C-Raf–membrane contacts. The region, res. 143–150, is indicated in blue. Inset images: representative orientations of RBD and CRD at the membrane. (c) POPS distribution at the membrane. The distribution is normalized to make the maximum 100%. K-Ras4B CD1 is centered at (0, 0); the horizontal displacement between the center of mass of K-Ras4B CD1 and that of C-Raf RBD is aligned to the x-axis. The last 500 ns of the trajectories were used in these analyses.

Figure 5

Configurations of the C-Raf–K-Ras4B complex at the membrane. The variables are defined as follows: D1, distance of K-Ras4B CD2, and D2, distance of CRD to membrane center, respectively. D, distance between the center of RBD–K-Ras4B^CD1 and membrane center. V1, vector connecting the center of K-Ras4B CD1 to center of the RBD. θ, cross angle between vector V1 and normal to the membrane surface. K-Ras4B CD1, CD2, RBD, CRD in cyan, blue, orange, and red, respectively. (a) Contour maps (scaled to max.) with variables D1 versus D2. (b) D versus θ. (c) Representative configurations for the four possible states of the C-Raf–K-Ras4B complex. +/− denotes whether the domain binds the membrane or not.

Figure 6

Tug of war between K-Ras4B and C-Raf CRD membrane interactions. (a) Schematic picture of steric/geometric limitations of the C-Raf–K-Ras4B complex in different states (here state 2; see Supporting Information, Figure S8 for state 1). (b) Potential of mean force, PMF, for K-Ras4B and CRD binding to the model membrane.