GDP-Loaded K-Ras Transiently Binds to Effector B-Raf RBD, Mirroring the Structure of the Active GTP-Loaded Complex.

The Ras-Raf protein association initiates the downstream mitogen-activated protein kinase (MAPK) signaling cascade. This interaction depends on the Ras-nucleotide ligand, whereby Raf preferentially binds to the active guanosine triphosphate (GTP)-loaded state over the guanosine diphosphate (GDP)-loaded state of Ras. Whether GDP-bound Ras can also form specific protein-protein interactions with Raf, however, remains unclear. Here, we characterize the interaction between human K-Ras in both nucleotide states and the Ras-binding domain (RBD) of B-Raf by solution NMR. Active K-Ras·GTP forms a tight, conformationally restricted complex with RBD, causing pronounced chemical shift perturbations at the binding interface consistent with existing cryo-EM and X-ray crystallography structures. Surprisingly, we detect specific binding between "inactive" wild-type K-Ras·GDP with RBD in the millimolar affinity range, whereby the oncogenic G12D mutant of K-Ras further strengthens this interaction. NMR relaxation dispersion and chemical exchange saturation transfer (CEST) experiments allow the detailed structural characterization of the transient K-Ras·GDP·RBD complex, along with the determination of the interaction affinity and kinetics. The results demonstrate that the transient K-Ras·GDP·RBD bound state closely resembles the active K-Ras·GTP·RBD complex, providing a quantitative understanding at the backbone 15N-level of the nucleotide-specific K-Ras-Raf interaction. These findings underscore the prospect of the highly adaptable GDP-bound state of K-Ras for both signaling and as a drug target.