Conformational mapping of GPCR activation: dynamic allosteric site discovery in V2R through MD-MSM and mutual information analysis
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Allostery governs the functional dynamics of proteins by regulating their conformational transitions. The development of allosteric modulators has emerged as a promising therapeutic strategy, leveraging their superior target specificity and reduced off-target effects compared to orthosteric compounds. A critical barrier in this field remains the identification of dynamic allosteric sites, which are often undetectable in conventional structural analyses due to their transient nature. To address this challenge, we established an integrative computational framework combining molecular dynamics (MD), Markov state modeling (MSM), and mutual information (MI) analysis to probe dynamic allosteric sites in the class A G protein-coupled receptor (GPCR) prototype, vasopressin V2 receptor (V2R). Through multi-replica MD simulations, we reconstructed the receptor's conformational landscape, which was statistically refined via MSM to resolve equilibrium populations and transition kinetics. Key mechanistic features of activation-related structural motifs were quantitatively characterized. Candidate allosteric sites were systematically ranked through MI-driven residue interaction network analysis, prioritizing pharmacologically targetable regions. This strategy revealed a novel dynamic allosteric site on the V2R intracellular interface, whose functional relevance was confirmed through structure-guided mutagenesis and BRET-based signaling assays. Our findings not only elucidate the allosteric activation mechanism of V2R at atomic resolution but also establish a conformation-aware platform for rational discovery of dynamic binding pockets, providing a transformative approach for GPCR-targeted drug discovery.
