De novo design of phospho-tyrosine peptide binders

Phosphorylation on tyrosine is a key step in many signaling pathways. Despite recent progress in de novo design of protein binders, there are no current methods for designing binders that recognize phosphorylated proteins and peptides; this is a challenging problem as phosphate groups are highly charged, and phosphorylation often occurs within unstructured regions. Here we introduce RoseTTAFold Diffusion 2 for Molecular Interfaces (RFD2-MI), a deep generative framework for the design of binders for protein, ligand, and covalently modified protein targets. We demonstrate the power and versatility of this method by designing binders for four critical phosphotyrosine sites on three clinically relevant targets: Cluster of Differentiation 3 (CD3ε), Epidermal Growth Factor Receptor (EGFR) and Insulin Receptor (INSR). Experimental characterization shows that the designs bind their phospho-tyrosine containing targets with affinities comparable to native binding sites and have negligible binding to non-phosphorylated targets or phosphopeptides with different sequences. X-ray crystal structures of generated binders to CD3ε and EGFR are very close to the design models, demonstrating the accuracy of the design approach. RFD2-MI provides a generalizable all-atom diffusion framework for probing and modulating phosphorylation-dependent signaling, and more generally, for developing research tools and targeted therapeutics against post-translationally modified proteins.