Discovering cryptic pocket opening and binding of a stimulant derivative in a vestibular site of the 5-HT3A receptor
Abstract
Ligand-gated ion channels propagate electrochemical signals in the nervous system. A diverse set of allosteric modulators including stimulants, anesthetics, and lipids regulate their function;however, structures of ligand-bound complexes can be difficult to capture by experimental methods, particularly when binding is dynamic or transient. Here, we used computational methods and electrophysiology to identify a possible bound state of a modulatory stimulant derivative in a cryptic vestibular pocket of a mammalian serotonin-3A receptor. Starting from a closed-pocket experimental structure, we first applied a molecular dynamics simulations-based goal-oriented adaptive sampling method to identify possible open-pocket conformations. To find plausible ligand-binding poses, we performed Boltzmann docking, which combines traditional docking with Markov state modeling, of the newly sampled conformations. Clustering and analysis of stability and accessibility of docked poses supported a preferred binding site; we further validated this site by mutagenesis and electrophysiology, suggesting a mechanism of potentiation by stabilizing intersubunit contacts. Given the pharmaceutical relevance of serotonin-3 receptors in emesis, psychiatric and gastrointestinal diseases, characterizing relatively unexplored modulatory sites such as these could open valuable avenues to understanding conformational cycling and designing state-dependent drugs.
