Studies On Interaction Mechanism Between G-Protein Coupled Receptors And Their Ligands Using Molecular Simulations

G protein coupled receptors (GPCRs), a large family of seven transmembrane domain receptors, are the target of approximate40%of all modern drugs. And they are only found in the eukaryotes and play the key role on some physiologic regulations, such as stress response, cardiovascular regulation, appetite control, immune function, glucose metabolism and other physiological responses. GPCRs were generally classified into six main families on basis of the sequence and structure similarity:class A (rhodopsin-like), class B (secretin receptor family), class C (metabotropic glutamate/pheromone), class D (fungal mating pheromone receptors), class E (cyclic AMP receptors) and class F (frizzled/smoothened). In recent years, the crystal structures of class A, B and F GPCRs are determined. Therefore, in this disseratation, the interaction mechanism between ligands and Class A, B and F GPCRs are studied by using molecular docking, moelcular dynamics (MD) simulations, free energy calculation dynamical network analysis and Markov state model. The main works in this dissertation include:1、For class A GPCR, we studied the conformational change of β2adrenergic receptor (P2AR) induced by different ligands. The simulation results demonstrate that agonist BI-167107can form hydrogen bonds with Ser2035.42, Ser2075.46and Asn2936.55more than the inverse agonist ICI118,551. The different binding modes of ligands further affect the conformation of β2AR. The energy landscape profiles the energy contour map of stable and dissociated conformation of Gas and Gβγ when different types of ligands bind to β2AR. It also shows the minimum energy pathway about the conformational change of Gas and Gβγ along the reaction coordinates. By using interactive essential dynamics analysis, the Gas and Gβγ domain of Gs protein is found to have the separated tendency when the inverse agonist ICI118,551binds to β2AR. The a5-helix has a relatively quick movement with respect to transmembrane segments of β2AR when the inverse agonist ICI118,551binds to β2AR. Besides, the analysis of the centroid distance of Gas and Gβγ shows that the Gas is separated from Gβγ during the MD simulations. These results not only can provide details about the different types of ligands induced conformational change of β2AR and Gs protein, but also supply more information for different efficacies of drug design of β2AR. 2、1μs molecular dynamics simulations on β2AR in complex with nucleotide-free Gs heterotrimer and inverse agonist ICI118,551is performed to study the detailed mechanism about the inverse agonist induced conformational change of β2AR from active to inactive state, as well as the implication of the status of the water channel with the active to inactive conformational change. The results show that inverse agonist ICI118,551can induce the conformation of (32AR to change from active conformation to inactive conformation. The water channel is opened gradually after about560ns in the1μs molecular dynamics simulations. The Markov state model (MSM) analysis further divides the energy contour into seven states. The state S1, S2and S5, which represent the active conformation of β2AR, show the water channel is in close state. The state S4and S6, which correspond to the intermediate state conformation of β2AR, indicate the water channel opens gradually. And the state S7, which matches with the inactive structure of β2AR, corresponds to the full open of the water channel. These results can provide useful information to understand molecular mechanism of the inverse agonist induced change of β2AR from active to inactive conformation and the role of water channel during the process of conformational change of β2AR.3、For class B GPCR, the escape pathway of antagonist in the dynamical pocket of corticotropin-releasing factor receptor1(CRF1R) is explored by molecular dynamics (MD) simulations, dynamical network analysis, random acceleration molecular dynamics (RAMD) simulations and adaptive biasing force (ABF). The results of dynamical network analysis show the TM7part of CRF1R has the strongest edges during MD simulations. And the bent part of TM7, which forms the V-shape pocket of CRF1R, is due to the Gly3567.50and strongest edges of TM7. The residue Asn2835.50, which has high hydrogen bonds occupancy during100ns MD simulations, plays an important role on the interaction with the antagonist in the pocket of CRF1R. The RAMD simulations identify three pathways (PW1, PW2and PW3) along which the antagonist can escape from the binding pocket of CRF1R. The PW3is proved to be the most likely escaped pathway of the antagonist CP-376395. The free energy along the PW3is calculated using ABF simulations. Two energy barriers are detected along the reaction coordinates. The residues Leu3236.49, Asn2835.50and Met2063.47form the steric hindrance for the first energy barrier. The residue His1993.40and Gln3557.49, which has high hydrogen bonds occupancy, constructs the strong interaction with the antagonist CP-376395for the second energy barrier. These results not only provid the information about the interaction mechanism between the antagonist CP-376395and CRF1R, but also supply the possible escaped pathway and free energy profile of the antagonist in the pocket of CRF1R for further drug design.4、For class F GPCR, the dynamical structural features of human smoothened receptor and binding mode of antagonist ly2940680are studied by molecular dynamics (MD) simulations, dynamic network analysis, metadynamics simulations and free energy calculation. The MD simulated results and dynamical network analysis show that the conserved KTXXXW motif in helix VIII has the strong interaction with the helix I. And the a-helical extension of TM6, which is stabilized by the hydrogen-bonding network, is detected as part of the ligand-binding pocket. The interactive analysis shows the N219of ECD linker domain and D384of the β-hairpin of ECL2form the hydrogen bonds with antagonist. The metadynamics results indicate the antagonist in SMO receptor has different energetic distributions by comparing with antagonist in water box. The adaptive biasing force (ABF) simulations show the antagonist need overcome about38kcal/mol energy barrier to leave binding pocket of SMO receptor. Besides, the results also suggest that the a-helical extension of TM6is an important domain for dissociated pathway of antagonist. All the results can not only profile the binding mechanism of the ligands in the different kinds of GPCRs, but also supply the useful information for the rational design of more potential small drug molecule bound to GPCRs.