Abstract
RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. 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. In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the 14-3-3 protomer bound to the BRAF C-terminal site and that key basic residues required for RBD-RAS binding are exposed. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the center of the RBD:14-3-3 interface have a dual function, first contributing to RAF autoinhibition and then to the full spectrum of RAS-RBD interactions.
Highlights
RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear
Using a mammalian cell expression system to isolate Bank under accession numbers PDB ID: 7MFD (BRAF) complexes, here we report the cryoelectron microscopy (cryo-EM) structures of two autoinhibited, monomeric BRAF complexes in which the RAS binding domain (RBD) is well-defined
As was reported for the ATP-analog-bound structures, the N- and C-lobes of the kinase domains (KD) in our ATP-free structures exhibited a closer orientation than is observed for the lobes of KDs bound to RAF inhibitors (Supplementary Fig. 5b, and Supplementary Table 2)[17,20,26,27]. These findings indicate that the ATP-binding pocket of the BRAF KD is stable in the “apo” nucleotide-free state and that while ATP-binding may be required to form the compact configuration of the N- and C-lobes as well as the autoinhibited BRAF conformation, these states can exist in the absence of bound ATP (Supplementary Fig. 5b, c, and Supplementary Tables 1, 2)
Summary
RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. We present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:[14-332] complexes, and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the [-3] protomer bound to the BRAF C-terminal site and that key basic residues required for RBD-RAS binding are exposed. Resolution of the RBD in our autoinhibited, monomeric BRAF structures provides key insights regarding how the orientation of the RBD allows access for RAS binding and how the interaction with RAS initiates the monomer-to-dimer transition required for RAF activation
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