Abstract
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.
Highlights
Melatonin (N-acetyl-5-methoxytryptamine, Figure 1, compound 1) is a neurohormone mainly synthesized by endocrine cells situated in the pineal gland following the circadian rhythm, with elevated levels at night
Intracellular loop 3 (ICL3, Gln219-Pro[227] in MT1 and Arg232-Leu[240] in MT2 receptors) and missing side chains were added with Modeller 9.21.27 One hundred models were generated for each structure, leaving residues adjacent to the reconstructed loop flexible to allow proper geometries (Arg[218] and Lys[228] for the MT1 receptor, Arg[231] and Cys[241] for the MT2 receptor), while the rest of the protein was kept frozen during the optimization of the spatial restraints
We investigated the feasibility of a lipophilic route for melatonin receptor ligands that can transit from the orthosteric binding site, located in the 7-TM bundle, to the membrane through a lateral channel between TM helices IV and V
Summary
Melatonin (N-acetyl-5-methoxytryptamine, Figure 1, compound 1) is a neurohormone mainly synthesized by endocrine cells situated in the pineal gland following the circadian rhythm, with elevated levels at night. Melatonin is described as a pleiotropic molecule with a multiplicity of effects[1] and a variety of cellular targets.[2] In mammals, modulation of several physiological and neuroendocrine functions occurs through activation of the two class A G-protein-coupled receptors (GPCRs) MT1 and MT2, for which melatonin shows subnanomolar binding affinity.[3,4] Activation of the two receptors is involved in the entrainment of circadian and seasonal rhythms and in regulation of the sleep-wake cycle,[5] as well as in a multitude of other physiological functions, comprising regulation of body temperature and hormone secretion, homeostasis of glucose secretion and of the cardiovascular system, and pain perception. Several melatonin receptor ligands have been synthesized, with distinct levels of intrinsic activity and receptor subtype selectivity,[8,9] and MT1/MT2 nonselective agonists have been approved for the treatment of insomnia, circadian rhythm disorders, and major depression
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