Discovering cryptic pocket opening and binding of a stimulant derivative in a vestibular site of the 5-HT3A receptor

Ligand-gated ion channels propagate electrochemical signals in the nervous system. A diverse set of 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, distinct from the orthosteric neurotransmitter binding site, 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. Significance5-HT3A receptors receive the chemical signals of excitatory neurotransmission across the synapse in the central and peripheral nervous systems, and are involved in conditions including emesis, pain, psychiatric disorders, drug abuse, and irritable bowel syndrome. Given their pharmaceutical importance, there is great interest in understanding how and where ligands interact with these receptors. A pocket facing the extracellular vestibule of this membrane protein has been proposed as a modulatory site, but it remains largely uncharacterized in the context of structural modeling or pharmacologically relevant ligands. Here, we are able to identify and investigate binding of a stimulant derivative, 4-bromoamphetamine, in this site by using an integrative computational and experimental approach that is able to account for conformational flexibility.