Raf-1 Cysteine-Rich Domain Increases the Affinity of K-Ras/Raf at the Membrane, Promoting MAPK Signaling

K-Ras4B preferentially activates Raf-1. The high-affinity interaction of Ras-binding domain (RBD) of Raf with Ras was solved, but the relative position of Raf's cysteine-rich domain (CRD) in the Ras/Raf complex at the membrane and key question of exactly how it affects Raf signaling are daunting. We show that CRD stably binds anionic membranes inserting a positively charged loop into the amphipathic interface. Importantly, when in complex with Ras/RBD, covalently connected CRD presents the same membrane interaction mechanism, with CRD locating at the space between the RBD and membrane. To date, CRD's role was viewed in terms of stabilizing Raf-membrane interaction. Our observations argue for a key role in reducing Ras/RBD fluctuations at the membrane, thereby increasing Ras/RBD affinity. Even without K-Ras, via CRD, Raf-1 can recruit to the membrane; however, by reducing the Ras/RBD fluctuations and enhancing Ras/RBD affinity at the membrane, CRD promotes Raf's activation and MAPK signaling over other pathways. Li et al. analyze the dynamic conformations of membrane-bound K-Ras4B in complex with the Ras-binding domain (RBD)-cysteine-rich domain (CRD) of Raf. The analysis suggests that K-Ras4B reduces Ras/RBD fluctuations at the membrane, thus increasing Ras/RBD affinity, and promoting Raf activation and MAPK signaling.