Mapping binding landscapes of Ras/effector interactions

Protein-protein interactions (PPIs) control a wide variety of biological activities in the cell. Our goal is to study evolution of PPIs that are involved in signaling through mapping of their binding landscapes. As a model PPI, we picked an interaction between a small GTPase Ras and its effector Nore1A. Ras is a molecular switch that binds with high affinity to various effectors in the GTP state but loses its affinity to effectors upon GTP hydrolysis. Several single mutations on Ras lock it in the “on” state and promote constant interactions with its effectors, resulting in cancer. Using state-of-the-art technology that utilizes Yeast Surface Display (YSD), deep sequencing, and data normalization, we study how binding landscapes of the Ras/Nore1A interaction change upon switching between different physiological states and due to oncogenic mutations, providing first insights on evolutionary paths of PPIs involved in signaling. For this purpose, we constructed a library of 40.000 single and double Nore1A mutants randomizing 15 binding interface positions. The Nore1A library was expressed on the surface of yeast cells, incubated with either Ras-GTP or Ras-GDP and sorted by FACS into four populations with high, wild-type, slightly lower, and very low affinities. Nearly ten million Nore1A mutants were incubated with Ras-GTP and Ras-GDP and were subjected to deep sequencing with the goal of identifying mutations important for preserving this interaction in various physiological and mutated Ras states. This research will greatly help our understanding of how signaling PPIs evolve and how oncogenic mutations perturb their binding landscapes.