Free-Energy Simulations Support a Lipophilic Binding Route for Melatonin Receptors

[Image: see text] The effects of the neurohormone melatonin are mediated by the activation of the GPCRs MT(1) and MT(2) in a variety of tissues. Crystal structures suggest ligand access to the orthosteric binding site of MT(1) and MT(2) receptors through a lateral channel between transmembrane (TM) helices IV and V. We investigated the feasibility of this lipophilic entry route for 2-iodomelatonin, a nonselective agonist with a slower dissociation rate from the MT(2) receptor, applying enhanced sampling simulations and free-energy calculations. 2-Iodomelatonin unbinding was investigated with steered molecular dynamics simulations which revealed different trajectories passing through the gap between TM helices IV and V for both receptors. For one of these unbinding trajectories from the MT(1) receptor, an umbrella-sampling protocol with path-collective variables provided a calculated energy barrier consistent with the experimental dissociation rate. The side-chain flexibility of Tyr5.38 was significantly different in the two receptor subtypes, as assessed by metadynamics simulations, and during ligand unbinding it frequently assumes an open conformation in the MT(1) but not in the MT(2) receptor, favoring 2-iodomelatonin egress. Taken together, our simulations are consistent with the possibility that the gap between TM IV and V is a way of connecting the orthosteric binding site and the membrane core for lipophilic melatonin receptor ligands. Our simulations also suggest that the open state of Tyr5.38 generates a small pocket on the surface of MT(1) receptor, which could participate in the recognition of MT(1)-selective ligands and may be exploited in the design of new selective compounds.