biostudies-literatureUnknownKim ARNCI NIH HHSNIAMS NIH HHSNIGMS NIH HHSProtein kinases orchestrate cellular processes through phosphorylation, yet the structural basis for their specific binding partner interactions remains largely unmapped. Here, we present a structure-guided atlas of the human and <i>Drosophila</i> kinome, built by applying a new interface-aware scoring framework (iLIS) to AlphaFold-Multimer predictions. The resulting atlas recapitulates hallmark sequence preferences, confirms previously reported and functionally related protein-protein interactions, and uncovers unrecognized docking interactions. Notably, our analysis predicts a potentially widespread docking motif on homeodomain transcription factors that mediates interactions with basophilic kinases. Furthermore, we map putative allosteric interaction hotspots across the kinome and provide proof-of-concept evidence that targeting these surfaces can inhibit kinase activity. Finally, we demonstrate the physiological utility of the atlas by identifying a novel regulatory mechanism between Sgg/GSK3 and Hnf4 that controls lipid metabolism <i>in vivo</i>. This resource provides a blueprint for dissecting signaling networks and for the rational design of docking-site-specific kinase modulators.bioRxiv : the preprint server for biology2025.10.10.681672https://www.ebi.ac.uk/biostudies/studies/S-EPMC12632555biostudies-literatureA Structure-Guided Kinase-Transcription Factor Interactome Atlas Reveals Docking Landscapes of the Kinome.PMC12632555R01 AR057352P41 GM132087R35 CA197588P01 CA117969P01 CA120964Johnson JLHu YPerrimon NKim ARCantley LCHuang KYaron-Barir TMWang KfalseA Structure-Guided Kinase-Transcription Factor Interactome Atlas Reveals Docking Landscapes of the Kinome.Protein kinases orchestrate cellular processes through phosphorylation, yet the structural basis for their specific binding partner interactions remains largely unmapped. Here, we present a structure-guided atlas of the human and <i>Drosophila</i> kinome, built by applying a new interface-aware scoring framework (iLIS) to AlphaFold-Multimer predictions. The resulting atlas recapitulates hallmark sequence preferences, confirms previously reported and functionally related protein-protein interactions, and uncovers unrecognized docking interactions. Notably, our analysis predicts a potentially widespread docking motif on homeodomain transcription factors that mediates interactions with basophilic kinases. Furthermore, we map putative allosteric interaction hotspots across the kinome and provide proof-of-concept evidence that targeting these surfaces can inhibit kinase activity. Finally, we demonstrate the physiological utility of the atlas by identifying a novel regulatory mechanism between Sgg/GSK3 and Hnf4 that controls lipid metabolism <i>in vivo</i>. This resource provides a blueprint for dissecting signaling networks and for the rational design of docking-site-specific kinase modulators.2025-01-01T00:00:00Z2025 Nov2026-06-10T03:13:32.005Z2026-06-10T03:07:44.35ZS-EPMC126325554127973610.1101/2025.10.10.681672