Protein-protein interactions (PPIs) are key drivers of cell signaling networks and represent important therapeutic targets in numerous physiological processes and pathologies. Despite their importance, interrogating and perturbing aberrant PPIs has proved challenging due to the nature of these interactions. The large surface contacts between proteins in addition to the duration of the interactions, remain difficult to examine. To begin to investigate PPIs and to develop general strategies to interrogate PPIs, we chose to use KRAS and its binding partners as an exemplar. The small GTPase KRAS is a central hub in cellular signaling networks that are responsible for proliferation and it is mutated in 20-30% of all cancers. Yet, there are no therapeutics on the market that directly target this protein. To further study KRAS and its mutant forms, including its binding domains and the biophysical properties of its ligands, we developed a generalizable platform to reprogram PPIs through rapid continuous evolution. This approach utilizes our previously designed split T7 RNA polymerase (RNAP) biosensor and the previously reported directed evolution platform phage assisted continuous evolution (PACE), to evolve a new PPI partner for the mutant KRAS G12D protein. Initially we utilized a native binding partner of KRAS, RAF, to develop and test the new PACE PPI-selection platform. Using our designed PACE selection platform, we show that proteins with an affinity for the KRAS mutant will outcompete other proteins that do not have affinity for the KRAS mutant. We also demonstrate that we can evolve proteins to gain activity for KRAS G12D by using a mutant form of RAF without affinity to KRAS G12D as the initial starting points in our evolution. Based on these results, we have designed a PPI evolution platform to evolve native protein binders for KRAS G12D, and we plan to evolve other small protein scaffolds for KRAS G12D using this platform.

Development and application of a continuous evolution system to program protein-protein interactions

2019 Spring National Meeting

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