Interpretable Machine Learning Model of Receptor Dynamics Reveals AT1R Allostery and a Negative Allosteric Modulator

Allosteric modulation of G protein–coupled receptors (GPCRs) offers major advantages in receptor selectivity and signaling control; yet systematic approaches to identify allosteric modulators, define their binding sites, and map the underlying allosteric networks remain limited. Current molecular dynamics (MD) and machine learning (ML)-based methods often rely on correlation-driven or black-box models that provide limited mechanistic insight. We developed an interpretable probabilistic framework that extracts residue-level dependencies from MD ensembles using Bayesian network modeling (BNM). By representing each residue through its local interaction energy, BNM identifies both local and long-range energetic couplings and maps the allosteric communication pathways linking the AngII binding site to the G-protein interface in the angiotensin II type 1 receptor (AT1R). To functionally prioritize these pathways, we integrated BNM with comprehensive mutational analysis, combining whole-receptor alanine mutagenesis data with exhaustive in silico deep mutational scanning to validate BNM-predicted hotspots. This approach recovered state-dependent allosteric communities, revealed residues in noncanonical regions that regulate Gα _q coupling and identified positions whose functional importance emerged only with specific, predicted substitutions, as well as highlighted a cryptic intracellular pocket enriched in communication hubs. Guided by these network-derived residues and pocket geometries, structure-based virtual screening identified a small, fragment-like molecule negative allosteric modulator (NAM) named Q2 that attenuates AngII-mediated Gα _q signaling. Mutational mapping supports Q2 binding adjacent to the G-protein interface, consistent with its mechanism of action. Together, these results establish a generalizable and interpretable framework for uncovering GPCR allosteric communication networks and discovering modulators that exploit these networks.