Thermodynamics of Ras/effector and Cdc42/effector interactions probed by isothermal titration calorimetry

Proliferation, differentiation, and morphology of eucaryotic cells is regul ated by a large network of signaling molecules, Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to d ifferent sets of effector proteins. Recognition of multiple effecters is im portant for communicating signals into different pathways, leading to the q uestion of how an individual GTPase achieves tight binding to diverse targe ts. To understand the observed specificity, detailed information about bind ing energetics is expected to complement the information gained from the th ree-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ra s subfamily with four different effecters and, additionally, the more dista ntly related Cdc42/WASP couple were quantified by means of isothermal titra tion calorimetry. The heat capacity changes upon complex formation were rat ionalized in light of the GTPase/effector complex structures. Changes in en thalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different, The energy profile of the Cdc42/WASP interaction is similar to R as/Ral guanine nucleotide dissociation stimulator, despite largely differen t structures and interface areas of the complexes. Water molecules in the i nterface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.