Mapping binding landscapes of Ras/effector interactions

Protein–protein interactions (PPIs) regulate a wide range of cellular activities and play essential roles in signaling pathways. Our goal is to investigate the evolutionary dynamics of PPIs by mapping their binding landscapes. As a model system, we focused on the interaction between the small GTPase Ras and its effector Nore1A. Ras functions as a molecular switch, binding effectors with high affinity in its GTP-bound state and losing affinity upon GTP hydrolysis. Single point mutations in Ras can lock it in the “on” state, promoting constitutive effector interactions that drive oncogenesis. Using a combination of Yeast Surface Display (YSD), deep sequencing, and quantitative data normalization, we explored how the Ras/Nore1A binding landscape changes under different physiological states and oncogenic mutations. To this end, we constructed a library of nearly 34,000 single and double Nore1A mutants by randomizing 14 binding interface residues. The library was expressed on yeast cell surfaces, incubated with either mutant Ras G12D or wild-type Ras in the presence of GTP or GDP, and sorted by FACS into four affinity-based populations: high, wild-type, moderately reduced, and very low. Nearly ten million Nore1A variants were subjected to deep sequencing, enabling the identification of optimized PPI variants and the mapping of binding hot- and cold-spots onto the crystallographic structure. This study provides novel insights into the evolutionary trajectories of signaling PPIs and reveals how oncogenic mutations reshape binding landscapes, thereby advancing our understanding of molecular mechanisms underlying cancer progression.