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.

Understanding how cell-penetrating peptides translocate across cell membranes is of great importance in biomedicine. In this paper, we study the interactions between polyarginines and asymmetric membranes by using coarse-grained molecular dynamics simulations. It is found that a peptide has the probability to penetrate through the membrane because of the transmembrane potential difference; however, it is difficult for a single peptide to spontaneously penetrate through the membrane while multiple peptides can translocate across membranes by pore-mediated processes. Further, we also provide insights into the transporting ability of polyarginines, and find that the peptide can transport hydrophobic as well as hydrophilic particles through membranes, where the translocation of a hydrophobic particle is easier than that of a hydrophilic one. The present study can help to better understand the interactions of the peptides with cell membranes and may give some new suggestions on the design of future nanomaterials for drug delivery.

Ras genes potently drive human cancers, with mutated proto-oncogene GTPase KRAS4B (K-Ras4B) being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the “holy grail” of present-day cancer therapy, and recent discoveries of small-molecule KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Since interactions with biological membranes are key for Ras function, Ras–lipid interactions have become a major focus, especially since such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysical and cell biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidyl inositol (4,5) phosphate (PIP2). We discovered that the β2 and β3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras–mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. These findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; insights that may inform the future design of therapeutic reagents targeting Ras activity.

Significance: Ras GTPases, KRas, HRas, and NRas control cell proliferation via the Ras/Raf/MEK/ERK pathway through a process in which Ras dimerization is required but not mechanistically understood. Here, we show that Raf-RBD promotes Ras dimerization resulting in strong allosteric linkages between Galectin-binding residues on Raf-RBD at the two extreme ends of the dimer. This suggests that the dimer of the Ras/Raf–RBD complex couples to Galectin dimers to form a multivalent signaling complex for synchronized activation of Raf kinase, providing a new framework to be tested in the mechanistic understanding of Ras/Raf signaling.