Unveiling Large-Scale Kinase-Centric Protein–Protein Interactions through a Knowledge-Informed Workflow

Protein phosphorylation regulates signaling, yet atomic-level substrate specificity remains elusive due to sparse structural data and phosphorylation-site-insensitive deep-learning predictors. Here we present a pipeline reformulating kinase–substrate modeling as a Bayesian inference problem. By integrating curated data sets and literature evidence parsed by Large Language Models, we converted diverse biological knowledge into structural restraints for the restraint-guided deep-learning model GRASP. For EGFR, BRAF and JNK1, we obtained 336 new phosphorylation-site-specific structure candidates refined by molecular dynamics. These models recapitulate known features, such as JNK1’s hydrophobic docking groove, and enabled a Virtual Position Scanning Peptide Array (V-PSPA) to map recognition patches and derive sequence preferences. Cross-referencing predicted interfaces with AlphaMissense pathogenicity scores reveal that the interaction types and distances to the catalytic pocket significantly influence pathogenicity scores. A comparison with clinical mutation data sets further connects pathogenic mutations to the kinase–substrate interface. This high-resolution, high-throughput pipeline can be broadly applicable to kinase specificity studies and general drug discovery.