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 (Ras^GTP) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras^GDP) 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 Ras^GDP. Most of our designed mutations narrow the gap between the affinity of Raf for Ras^GTP and Ras^GDP, producing the desired shift in binding specificity towards Ras^GDP. A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras^GDP. The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras^GDP bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras^GDP Raf mutant complex is found in a conformation similar to that of Ras^GTP and not Ras^GDP. Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras^GTP is likely to explain the natural low affinity of Raf and other Ras effectors to Ras^GDP. 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.