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

The effects of the neurohormone melatonin are mediated by the activation of the GPCRs MT₁ and MT₂ in a variety of tissues. Crystal structures suggest ligand access to the orthosteric binding site of MT₁ and MT₂ 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 MT₂ 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 MT₁ 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 MT₁ but not in the MT₂ 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 MT₁ receptor, which could participate in the recognition of MT₁-selective ligands and may be exploited in the design of new selective compounds.