Molecular Simulations Study On The Cooperativity Mechanism Of Ligands In Functional Regulation Of Classes A And F GPCRs

The superfamily of G protein-coupled receptors(GPCRs)is a class of membrane proteins with seven transmembrane helices.GPCRs widely expressed in tissues and involving in a variety of physiological processes.GPCRs have been the most important drug targets due to their substantial involvement in human physiology,there are more than 30% of drugs that target GPCRs in all marketed drugs approved by the US Food and Drug Administration(FDA).The number of GPCRs targeted by approved drugs accounts for ~16% of more than 800 GPCRs,accordingly,there are still huge spaces and challenges in drug discovery and design targeting GPCRs.In addition to classical orthosteric drugs,allosteric modulators of GPCRs have unique advantages as drugs,including high selectivity and low toxicity,biased signalling and probe dependence.The allosteric modulation of GPCRs provides the diversity of protein function regulation and has become an important field in the development of drugs.There are several allosteric sites on GPCRs revealed by the crystal structures of complexes of GPCRs and allosteric regulators,indicating that allosteric sites likely span the surface of GPCRs.Functional assays and radioligand binding assays reveal cooperativity between the small molecule modulators sitting at different sites of GPCRs,including functional cooperativity and binding cooperativity,which effectively regulate the function of GPCRs.The crystal structures of GPCRs and small molecule modulators that bind at different sites provide a structural basis for understanding the detailed receptor-ligand interactions and allosteric cooperativity mechanism.The knowledge of the mechanisms about allosteric modulation of GPCRs and cooperativity between different sites has realistic instructive significance for structure-based drug discovery.However,the crystal structures resolved under specific experimental conditions are static,which limit our understanding of these mechanisms.The computer-assisted methods are the effective means to reveal the allosteric regulatory mechanisms of GPCRs,the detailed interactions of receptor-ligand,and mechanism of cooperativity between different sites.The class A GPCRs account for more than 85% of the GPCRs superfamily,and are currently the most studied GPCRs receptor family.There are diverse allosteric sites in class A GPCRs,even exists more than one allosteric sites on one receptor,such as the free fatty acid 1 receptor(GPR40).The partial agonist binding site and AgoPAM binding site of GPR40 where exist positive binding cooperativity and functional cooperativity;the orthosteric inhibitor and allosteric inhibitor of C5 a anaphylatoxin chemotactic receptor 1(C5aR1)together can stabilize the receptor,existing positive functional cooperativity;the presence of orthosteric inhibitor of CC chemokine receptor 2(CCR2)increases the total binding of allosteric inhibitor sitting in intracellular allosteric site,existing positive binding cooperativity.The class F GPCRs of GPCRs superfamily is the least studied.Vismodegib,a marketed drug,is the allosteric inhibitor of the smoothened receptor(Smo),allosterically inhibits the Smo activation and prevents cholesterol binding,existing negative binding cooperativity.The mechanisms of cooperativity between the different sites of receptors are currently unclear.We combined multiple accelerated molecular dynamics simulation methods to explore the detailed interaction between the receptor and ligand of allosteric sites,revealing the mechanism of cooperativity between different sites.This not only provides an understanding of the cooperativity mechanisms of modulators at different sites of GPCRs at the atomic level,but also has important implications for structure-based drug discovery and design.Our results suggest that the induced-fit conformational coupling between partial agonist and AgoPAM binding sites of GPR40 is bidirectional,and the two sites present positive binding cooperativity,the conformational dynamics of TM3,TM4 and TM5 are crucial in coordinating the conformational changes of the two sites.The orthosteric inhibitor and the allosteric inhibitor of C5aR1 can stabilize the extracellular side of TM5 and TM6,also can stabilize the intracellular side of TM7 by stabilizing allosteric site of sodium ion.The orthosteric inhibitor of CCR2 limits the movement of the extracellular side of TM6 by stabilizing the extracellular side of TM7,that stabilizes the intracellular side of TM6 that takes part in the binding of allosteric inhibitor.Because of the highly conserved prolines on TM5 and TM6 of class A GPCRs,giving the inherent flexibility of these helices,and enabling to take various conformational states;the conserved structural motifs on transmembrane helices play roles in triggering synergistic conformational changes between helices.Hence,the allosteric cooperativity between different sites of class A GPCRs is universal,and the mechanism of cooperativity is likely to be similar.The vismodegib binding with Smo induces shifts of TM5,TM6 and TM7,the movement of TM6 leads to synergistic conformational changes between the allosteric site and the orthosteric site,that results in the orthosteric pocket being occupied by extracellular extension of TM6 and ECL3;the movements of TM5 to TM7 stabilize the ICLs in inactive state.We revealed the significant conformational changes and difference of GPCRs binding with different ligands or their combinations,including the conformational states that not found in crystal structures,conformational changes of binding sites,and remolding of the binding mode of ligands.As in the GPR40,one of the sites bound induces rearrangement of TM4 and TM5,then leading to conformational change of the other site;while in the GPR40-apo,the entrances of both the sites are occluded.The similar situation takes place in the simulations of C5aR1,the allosteric site of C5aR-apo collapses,on the contrary,the allosteric site is maintained by binding of orthosteric ligand.This suggests that it is more reasonable to consider the remote effects of other site on the site in structure-based drug screening or design.The conformational states of the allosteric site in CCR2 significantly change in presence and absence of orthosteric inhibitor,that provides the structural information for screening novel skeleton compounds.The binding mode of vismodegib in Smo changes during the simulation,the new subsite can be considered in structural modification.Moreover,the significant conformational changes that occur during the simulation also suggest that the flexibility of the receptor should be fully considered when performing structure-based drug designing or screening,and the molecular dynamics simulation provides a reasonable and effective method.