Identification of an Unexplored Dynamic Allosteric Site on the Activation Pathway of the Vasopressin V2 Receptor

Allostery governs the functional dynamics of proteins
by regulating
their conformational transitions. A major challenge lies in identifying
dynamic allosteric sites, which are often not discernible from static
structural data. Here, we developed an integrative computational framework
combining molecular dynamics (MD) simulations, Markov state modeling
(MSM), and mutual information (MI) analysis to investigate the vasopressin
V2 receptor (V2R). Through multiple-replica MD simulations, we reconstructed
the receptor’s conformational landscape, which was statistically
refined using MSM to determine equilibrium populations and transition
kinetics. Key structural motifs associated with activation were quantitatively
characterized. Candidate allosteric sites were systematically prioritized
through MI-based residue interaction network analysis, highlighting
pharmacologically targetable regions. Our methodology uncovered an
unexplored dynamic allosteric site on the V2R intracellular interface,
whose functional relevance was confirmed through structure-guided
mutagenesis and BRET-based signaling assays. This approach establishes
a conformation-aware platform for detecting dynamic binding pockets,
providing a transformative approach for G protein-coupled receptor
(GPCR)-targeted drug discovery.