Massively parallel measurement of protein–protein interactions by sequencing using MP3-seq
Alexandr Baryshev, Alyssa La Fleur, Benjamin Groves, Cirstyn Michel, David Baker, Ajasja Ljubetič, Georg Seelig
Abstract
Protein–protein interactions (PPIs) regulate many cellular processes and engineered PPIs have cell and gene therapy applications. Here, we introduce massively parallel PPI measurement by sequencing (MP3-seq), an easy-to-use and highly scalable yeast two-hybrid approach for measuring PPIs. In MP3-seq, DNA barcodes are associated with specific protein pairs and barcode enrichment can be read by sequencing to provide a direct measure of interaction strength. We show that MP3-seq is highly quantitative and scales to over 100,000 interactions. We apply MP3-seq to characterize interactions between families of rationally designed heterodimers and to investigate elements conferring specificity to coiled-coil interactions. Lastly, we predict coiled heterodimer structures using AlphaFold-Multimer (AF-M) and train linear models on physics-based energy terms to predict MP3-seq values. We find that AF-M-based models could be valuable for prescreening interactions but experimentally measuring interactions remains necessary to rank their strengths quantitatively. A method called massively parallel PPI measurement by sequencing (MP3-seq) is developed for measuring protein–protein interactions at scale. MP3-seq uses DNA barcodes that are associated with specific protein pairs and provides a quantitative measure of interaction strength. Interactions between rationally designed heterodimers and elements conferring interaction specificity were investigated using MP3-seq.