What Makes Ras an Efficient Molecular Switch: A Computational, Biophysical, and Structural Study of Ras-GDP Interactions with Mutants of Raf

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (RasGTP) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (RasGDP) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with RasGDP. Most of our designed mutations narrow the gap between the affinity of Raf for RasGTP and RasGDP, producing the desired shift in binding specificity towards RasGDP. A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards RasGDP. The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of RasGDP bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the RasGDP·Raf mutant complex is found in a conformation similar to that of RasGTP and not RasGDP. Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in RasGTP is likely to explain the natural low affinity of Raf and other Ras effectors to RasGDP. Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch.