A unifying feature of the RAS superfamily is a functionally conserved GTPase cycle that proteins use to transition between active and inactive states. Here, we demonstrate that active site autophosphorylation of some small GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, thus altering the on/off switch that forms the basis for their signaling functions. Using x-ray crystallography, nuclear magnetic resonance spectroscopy, biolayer interferometry binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II domain and that autophosphorylation promotes nucleotide exchange by opening of the active site and extraction of the stabilizing Mg. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from the non-phosphorylated counterpart.

An unresolved issue in RAF kinase signaling is how binding of autoinhibited RAF monomers to activated RAS initiates the conformational changes required to form active RAF dimers. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. The RAS binding domain (RBD) of BRAF is resolved in the autoinhibited structures, and we find that neither MEK nor ATP binding is required to stabilize the autoinhibited complexes. Notably, the RBD was found to interact extensively with the 14-3-3 protomer bound to the BRAF C-terminal site. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the 14-3-3 interface have a dual function, first stabilizing RBD orientation in the autoinhibited state and then contributing to full RAS contact.