D1 Agonist and D2 Antagonist Dual Effect of SPD teraction between motion R Rt FIGURE 10 Signal transduction model of the DI receptor by an SPD-like agonist R represents the dynamic state, Rt, and Rt2 are transitional states before receptor activation, and r*refers to the activated state ready to couple with intracellular G-proteins Further nestling of SPD into the binding cavity brings about complexes, all simulated in a lipid bilayer membrane a dramatic outward movement toward lipid in TM6 and TM7 Distribution of the electrostatic potentials on the molecular (Rt2 state). Through this induced conformational shift, the surface and the conformational"open-closed"transition of receptor is activated and couples with the a-subunit of the the Dl and D2 receptors were addressed by the MD intracellular G-protein(R* state), transferring extracellular simulations. The K-167-_EL-2-E-302_EL-3 salt bridge in the signals to the intracellular G-protein. This stepwise signal DI receptor plays an important role in the conformational transduction model was supported by the currently accepted change of extracellular domain and in the binding of SPD postulate about the activation of GPCRs; that is, GPCRs The binding of Spd to the DI receptor causes a structural exist in an equilibrium between two interchangeable confor- relaxation, which is an early step in the activation of GPCRs mational states, namely a dynamic R state and an activated Major and significant structural differences were seen in the R* state(40, 41), and there should be considerable confor- TM6 and TM7 domains in the DI-SPD complex. In par- mational movement in TM6 and TM7 after agonist bindil g ticular, TM6 exhibits the most significant motion during d (17,19,52-54) receptor activation a detailed molecular level mechanism of the di agonistic Significance to future drug discovery targeting the and D2 antagonistic action of SPD has been delineated. Our D1/D2 receptors models indicate that the agonistic binding site in di and the ite in d2 shar DRs remain attractive and challenging drug targets when region in the structurally aligned receptors. However, SPD searching for potential therapeutics of psychological diseases takes on two different bioactive conformations, one in the (4, 41). Our demonstration of a potential molecular mecha- agonistic complex with DI and the other in the antagonistic nism for the dual function of SpD as an agonist of Dl and an complexes with D2. The structural determinants for the ntagonist of D2 provides practical guidance in the design of pharmacological specificity of dual action of SPD were novel lead compounds. We already have made some prom- uncovered from the modeling and simulations. Combine ing progress along these lines via our screening for novel with all the available experimental data, it can be concluded SPD-like compounds combined with experimental testing that residues H-6.55, W-7.40, and W-7.43 must be structural (W. Fu, W. Zhu, and H Jiang, unpublished data). Our com- determinants for differentiating the pharmacological dual putational results suggest that any small compound able to specificities of SPD for the DI and D2 receptors. Finally,a ack well with W-7.40 and W-7.43 in the DI receptor and potential signal transductic pack with H-6.55 in D2 may exhibit dual activity against the was proposed. Further mutagenesis and biophysical exper- I and D2 receptors, as does SPD iments are needed to test and improve our dual action echanism of SPD and stepwise signal transduc CONCLUSIONS Our results have already been used in database searching to identify new candidate compounds with dual actions We report herein four 10-ns MD simulations of the un- based on our identified DI agonist and D2 antagonist confor liganded DI and D2 receptors and DI-SPD and D2-SPD mations Several molecular leads for the eventual treatment Biophysical Joumal 93(5)1431-1441Further nestling of SPD into the binding cavity brings about a dramatic outward movement toward lipid in TM6 and TM7 (Rt2 state). Through this induced conformational shift, the receptor is activated and couples with the a-subunit of the intracellular G-protein (R* state), transferring extracellular signals to the intracellular G-protein. This stepwise signal transduction model was supported by the currently accepted postulate about the activation of GPCRs; that is, GPCRs exist in an equilibrium between two interchangeable confor￾mational states, namely a dynamic R state and an activated R* state (40,41), and there should be considerable confor￾mational movement in TM6 and TM7 after agonist binding (17,19,52–54). Significance to future drug discovery targeting the D1/D2 receptors DRs remain attractive and challenging drug targets when searching for potential therapeutics of psychological diseases (4,41). Our demonstration of a potential molecular mecha￾nism for the dual function of SPD as an agonist of D1 and an antagonist of D2 provides practical guidance in the design of novel lead compounds. We already have made some prom￾ising progress along these lines via our screening for novel SPD-like compounds combined with experimental testing (W. Fu, W. Zhu, and H. Jiang, unpublished data). Our com￾putational results suggest that any small compound able to pack well with W-7.40 and W-7.43 in the D1 receptor and pack with H-6.55 in D2 may exhibit dual activity against the D1 and D2 receptors, as does SPD. CONCLUSIONS We report herein four 10-ns MD simulations of the un￾liganded D1 and D2 receptors and D1-SPD and D2-SPD complexes, all simulated in a lipid bilayer membrane. Distribution of the electrostatic potentials on the molecular surface and the conformational ‘‘open-closed’’ transition of the D1 and D2 receptors were addressed by the MD simulations. The K-167_EL-2-E-302_EL-3 salt bridge in the D1 receptor plays an important role in the conformational change of extracellular domain and in the binding of SPD. The binding of SPD to the D1 receptor causes a structural relaxation, which is an early step in the activation of GPCRs. Major and significant structural differences were seen in the TM6 and TM7 domains in the D1-SPD complex. In par￾ticular, TM6 exhibits the most significant motion during D1 receptor activation. A detailed molecular level mechanism of the D1 agonistic and D2 antagonistic action of SPD has been delineated. Our models indicate that the agonistic binding site in D1 and the antagonistic binding site in D2 share a common binding region in the structurally aligned receptors. However, SPD takes on two different bioactive conformations, one in the agonistic complex with D1 and the other in the antagonistic complexes with D2. The structural determinants for the pharmacological specificity of dual action of SPD were uncovered from the modeling and simulations. Combined with all the available experimental data, it can be concluded that residues H-6.55, W-7.40, and W-7.43 must be structural determinants for differentiating the pharmacological dual specificities of SPD for the D1 and D2 receptors. Finally, a potential signal transduction mechanism of the D1 receptor was proposed. Further mutagenesis and biophysical exper￾iments are needed to test and improve our dual action mechanism of SPD and stepwise signal transduction model. Our results have already been used in database searching to identify new candidate compounds with dual actions based on our identified D1 agonist and D2 antagonist confor￾mations. Several molecular leads for the eventual treatment FIGURE 10 Signal transduction model of the D1 receptor by an SPD-like agonist. R represents the dynamic state, Rt1 and Rt2 are transitional states before receptor activation, and R* refers to the activated state ready to couple with intracellular G-proteins. D1 Agonist and D2 Antagonist Dual Effect of SPD 1439 Biophysical Journal 93(5) 1431–1441