Structural Basis Of The Ligand Binding And Signaling Mechanism Of Melatonin Receptors

G protein-coupled receptors(GPCRs)constitute the largest superfamily of cell membrane receptors and regulate numerous physiological processes in almost all the human tissues and organs.The malfunction of GPCRs often results in many diseases,such as diabetes,Alzheimer disease,obesity,cardiovascular disease,inflammation,and cancer.As a result,GPCRs have been of long-standing interest as pharmacological targets.Of the 826 human GPCRs,approximately 350 non-olfactory members are regarded as druggable and 165 of them are validated drug targets,and latest statistical data indicate that 527 drugs(～34%of all drugs approved by the US Food and Drug Administration)act on GPCRs.Structure-based drug design is becoming the most efficient way,and the advances in GPCRs structural biology over the past two decades have propelled the improvement of novel drug discovery.Therefore,it is essential to understand and uncover the mechanisms of GPCR activities and signal transduction pathways.Human MT1(melatonin receptor type 1)and MT2(melatonin receptor type 2),which are class A GPCRs,have high affinity for the natural ligand melatonin,and regulate circadian rhythms,sleep,reproduction,nervous system functions,immune functions,cardiovascular physiology and glucose homeostasis.Therefore,MTs have been regarded as important clinical targets of insomnia,mood disorders,cancer,type 2 diabetes,and autoimmune diseases.Despite the high sequence similarity,differences in the signaling capacity and distribution of MT1 and MT2 have been documented.The two receptor types activate Gi proteins and MT1 couples additionally to Gq proteins;MT1 is expressed in the Locus Coeruleus and lateral hypothalamus,but MT2 is mainly located in the reticular thalamus.The signaling variation and localization difference probably result in the distinct in vivo functions of MT1 and MT2.Given the distinct physiological roles of MT1 and MT2,obtaining MTs subtype selective ligands is highly desirable.However,most of the drugs on the market or under clinical evaluation are nonselective,and developing subtype-selective drugs,especially MT1-selective drugs,is very difficult.The reason partially lies in the lack of precise structures providing information on the orthosteric ligand pocket.In this study,we determined five cryo-EM structures of human MTs-Gi signaling complexes:four structures of the MT1-Gi complexes,either with non-selective agonist ramelteon and 2-iodomeltonin at global resolution of 3.3 and 3.1(?),or with MT1selective agonist 5-HEAT and MTi-partial agonist UCM871 at global resolution of 3.7(?) and 3.9 (?),respectively,and one structure of the human MT2-Gi complex with the agonist ramelteon at a global resolution of 3.4 (?).Firstly,in both the MT1 and MT2 structures,the ligand of 2-iodomelatonin or ramelteon binds at the orthosteric pocket,and structural analysis indicate that the only entrance to the orthosteric binding site in the active state is the previously identified"lateral channel" between TM4 and TM5.Comparison of the orthosteric pocket from active and inactive structures revealed that the pocket in the active structures is more constricted in the central part.Moreover,the N4.60-Y5.38-H5.46 motif lining the solvent channel presents very different conformations,which results in a varied configuration of the active pocket.Specifically,conformational change of Y1875.38 in MT1 constricts the diameter of the channel entrance.In MT2,the corresponding residue Y2005.38 takes opposite conformational changes,which widens the entrance of the channel in active MT2 structure.In addition,the rearrangement of His5.46 induces the formation of"longitudinal channel" in both active MT1 and MT2.Molecular docking results indicated that the distinct conformation of Y5.38 is an important determinant for MT1selective ligands and the difference between the "longitudinal channel" of MTs is of great value for MT1-selective bitopic agonist design.Secondly,Structural comparison demonstrated that MTs activation mediated through classical motif and unique microswitches.So in contrast to other Gi-coupled receptors,MTs exhibit a large outward movement of TM6,which is considered a specific feature of Gs-coupled receptors.Therefore,the selectivity for the Gi and Gs coupling is not solely determined by the extent of TM6 movement.Accordingly,the Gαi presents a conformation deviating from those in other GPCR-Gi complexes.Comparison of the selective agonist and unselective agonist bound MT1-Gi structures revealed that the residues surrounding the entrance of the solvent channel take significant conformational changes,and the structural plasticity of the entrance of the solvent channel supports selective ligand recognized by MT1.In addition,it seems that the particular conformational state of ECL2 is critical for MTs activation,and the effects of 5-HEAT and UCM871 on solvent channel entrance and ECL2 may account for their selective and partial agonism.In summary,our results provide new clues for designing new chemotype and subtype-selective MT1 or MT2 melatonergic drugs,and further insights into understanding the molecular mechanisms underlying MTs activation and G protein coupling mechanism.