Mapping binding landscapes of Ras/effector interactions.

TOMAZINI, Atilio ¹

Vol 2, 2024 - 315621

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Abstract

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 mutant RAS G12D or wild type RAS in presence of GTP or GDP and then sorted by FACS into four affinity Nore1A-RAS populations: high, wild-type, slightly lower, and very low. Nearly ten million Nore1A mutants were subjected to deep sequencing with the goal of identify the more optimized PPIs targets and mapping the hot and cold spots into the crystallographic structure. This study will significantly advance our knowledge of how signaling PPIs evolve and how oncogenic mutations alter their binding environments.

Institutions

¹ The Hebrew University of Jerusalem

Track

18. Protein Structure and Conformation

Keywords

Protein-protein interactions (PPIs)

Ras oncogene

Protein Engineering

Protein Design