Raf promotes dimerization of the Ras G‐domain with increased allosteric connections

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Dimerization is a critical regulatory step throughout the MAPK signaling pathway. While dimerization of the Raf kinase domain is widely accepted and occurs even in the absence of Ras, Ras dimerization has been contested for several decades, but has been recently been shown to be essential for Raf activation. Our work shows that the Ras binding domain of Raf (Raf-RBD) promotes robust Ras dimerization on supported lipid bilayers and, to a lesser extent, in solution as confirmed via size-exclusion chromatography coupled to small-angle x-ray scattering. Community network analyses of molecular dynamics simulations of the dimer of the Ras/Raf-RBD complex reveal extensive allosteric connections unifying Raf-RBD into a single allosteric network and bridging the two Raf-RBD D113 residues, located within the predicted galectin scaffold protein binding site and 85 Å apart, at opposite ends of the complex. Our findings suggest that Ras/Raf-RBD binding and dimerization occur simultaneously at the membrane to yield a high-affinity signaling complex. We propose a higher order macromolecular assembly comprising Ras/Raf/galectin complexes for tightly regulated and synchronized signaling through the MAPK pathway.