The Journal of Physical Chemistry B Article and after ligand binding, the separation tor behaviors before triple mechanism of A-SPD. Of the 18 interacting trans- detailed understanding of dopamine re etween the centroid of membrane residues with I-SPD, 9 were conserved in all atoms CZ, NHl, NH2 in R3.50 and that of atoms CD, oEl, complexes and all 18 residues in both D2R and D3R were OE2 in E6.30 termed N-o distance"( Figure S3)was strictly conserved as revealed by sequence alignment in binding onitored and was chosen as an indicator of ionic lock pocket(Figure Sg). Their binding pes were different (Figure Sd-f) Specifically, in D2R and D3R, the large binding The ionic lock formed between R3. 50 in the E/DRY motif pocket 1 located between TM3, TMS, TM6, and TM7 was and E6.30 was proposed to have an essential role in maintaining slightly narrower than the corresponding binding pocket 1 in GPCRs in an inactive state. In the simulation of unliganded DIR; such narrowness was presumably resulted from the D2R, the N-o distance between R3. 50 and E6. 30 was stably relative bulkiness of the imidazole side chain H6.55 in D2R and formed a stable ionic lock with E6. 30 during MD simulation. Additionally, there was also a significant difference in ae onfined below 3. 8 A(Figure 3e); it indicated that R3. 50 D3R with respect to the amide side chain of N6.55 in DIR However, in the case of the 1-SPD-bound D2R, the orientation between Y7. 43 of D2R and D3R and w7. 43 of D1 orresponding N-o distance fluctuated between 3 and 7 A, in which y7. 43 pointed toward extracellular side, while w7.4 indicating that the ionic lock slightly fluctuated upon antagonist was directed toward cytoplasmic side. As a result of such binding. Similar to the unliganded D2R system, N-O distance difference, the binding pocket 2 of both D2R and D3R were of unliganded D3R was well conserved at ca. 3. 8 A within the separated from pocket 1, whereas in DiR, binding pocket 2 was first 38 ns of the simulation, and then it decreased to 3 A after merged with pocket 1. Previous efforts to rationalize the 38 ns, which signified a reinforcement of the ionic lock. While structural basis of selectivity have also focused on binding for the I-SPD bound D3 receptor, the ionic lock ruptured at ca. regions that are not conserved among DR subfamily 26 ns, the corresponding N-O distance even upsurged to sa primary attention being given to ECL2. 45+6 In DIR, C186, (Figure 3f). In terms of unliganded D2R and D3R, the D187, and $188 of ECL2 were found to be in contact with I- important feature in unliganded DiR simulation is that no ionic SPD with their counterparts in D2R and D3R being C182 lock is established( Figure 3d), but the distance in the TM3 1183, 1184 and C181, S182, 1183, respectively TM6 at the intracellular ends decreased to ca 12 A. In contrast Functionally relevant interactions between I-SPD and DIR, the corresponding distance kept almost constant(16-20 A) D2R, and D3R were then investigated to understand the the simulation of I-SPD-bound DIR, showing the important agonistic and antagonistic mechanism of I-SPD. Hydrogen of supports bonding, electrostatic (red dashed lines), and hydrophobic the point that the breakage of ionic lock is one of the features in interactions were observed as major contributions to I-SPD the active dir binding. As a common binding Functionally Relevant Interaction between /-SPD and protonated nitrogen atom to form electrostatic attraction with DIR, D2R/D3R. The predicted binding energies of I-SPD for negatively charged D3. 32 in DIR, D2R, and D3R(Figure 5a- DIR, D2R, and D3R are very well correlated with the c), such a common interaction was in well agreement with the experimental values as shown in Table 1 and Figure 4 fact that D3. 32 plays an essential role in ligand binding to aminergic GPCRs. 45 Table 1. Predicted and Experimental Binding Energies of 1- Despite the common binding features, differences in the SPD with DiR, D2R, and D3R binding interactions of I-SPD with all three receptors were dicted binding experimental binding energy remarkable. In the I-SPD-DiR complex, the binding orientation of I-SPD with DiR was reversed as compared to DIR 11.00 the binding orientations of 1-SPD with D2R and D3R(Figure 60 Sh). Given that the structure of I-SpD has one substructure of dopamine resided on both ends, this reversion of orientation was reasonable (Scheme 1). In addition, this adjustment of binding orientation of I-SPD with DiR was optimal for the formation of two hydrogen bonds(d ring hydroxyl group D OH with side chain of N6.55 and A ring hydroxyl group A-OH with D175 of r2=0.95 interactions were critical for the binding affinity of I-SPD with DIR as experimental results showed that simultaneous substitution of A-OH and d-oh by methoxyl groups resulted in monosubstitution of either A-OH or D-Oh by a methoxyl Experimental binding group resulted in a 6-fold loss of binding affinity(Table S3, (kalmo) entries 1 and 4). This binding mode is further supported by 4.Correlation between predicted binding energy and site-directed mutagenesis in B2AR in which N6.55 was mental binding energy demonstrated to be involved in a hydrogen bonding to the p. Oh group of adrenaline. #-49Furthermore, Manivet and co- Although there are deviations between the experimental value workers showed that N6.55 in the 5-HT2B receptor is involved (-10.80,-9.60, and-1066 kcal/mol)>and predicted data, in direct or indirect 5-HT binding the general trend is that l-SPD binds to DiR and D3R more In I-SPD-D2R and 1-SPD-D3R complexes, the overall strong ly than to D2R interaction landscape is similar to each other but with the DIR, D2R, or of -spd reversed relative to that in dir and d3r were initially compared ng at elucidating the previously mentioned. In both I-SPD-D2R and I-SPD-D3R, the 81 dx. dolora/o.021/p30492351 Phys. Chem. B2012116,8121-813detailed understanding of dopamine receptor behaviors before and after ligand binding, the separation between the centroid of atoms CZ, NH1, NH2 in R3.50 and that of atoms CD, OE1, OE2 in E6.30 termed “N−O distance” (Figure S3) was monitored and was chosen as an indicator of ionic lock formation. The ionic lock formed between R3.50 in the E/DRY motif and E6.30 was proposed to have an essential role in maintaining GPCRs in an inactive state. In the simulation of unliganded D2R, the N−O distance between R3.50 and E6.30 was stably confined below 3.8 Å (Figure 3e); it indicated that R3.50 formed a stable ionic lock with E6.30 during MD simulation. However, in the case of the l-SPD-bound D2R, the corresponding N−O distance fluctuated between 3 and 7 Å, indicating that the ionic lock slightly fluctuated upon antagonist binding. Similar to the unliganded D2R system, N−O distance of unliganded D3R was well conserved at ca. 3.8 Å within the first 38 ns of the simulation, and then it decreased to 3 Å after 38 ns, which signified a reinforcement of the ionic lock. While for the l-SPD bound D3 receptor, the ionic lock ruptured at ca. 26 ns, the corresponding N−O distance even upsurged to 5 Å (Figure 3f). In terms of unliganded D2R and D3R, the important feature in unliganded D1R simulation is that no ionic lock is established (Figure 3d), but the distance in the TM3− TM6 at the intracellular ends decreased to ca. 12 Å. In contrast, the corresponding distance kept almost constant (16−20 Å) in the simulation of l-SPD-bound D1R, showing the important role of l-SPD in stabilizing the active state of D1R. It supports the point that the breakage of ionic lock is one of the features in the active D1R. Functionally Relevant Interaction between l-SPD and D1R, D2R/D3R. The predicted binding energies of l-SPD for D1R, D2R, and D3R are very well correlated with the experimental values as shown in Table 1 and Figure 4. Although there are deviations between the experimental value (−10.80, −9.60, and −10.66 kcal/mol)15 and predicted data, the general trend is that l-SPD binds to D1R and D3R more strongly than to D2R. The differences in binding pocket lining among D1R, D2R, and D3R were initially compared aiming at elucidating the triple mechanism of l-SPD. Of the 18 interacting trans￾membrane residues with l-SPD, 9 were conserved in all complexes and all 18 residues in both D2R and D3R were strictly conserved as revealed by sequence alignment in binding pocket (Figure 5g). Their binding pocket shapes were different (Figure 5d−f). Specifically, in D2R and D3R, the large binding pocket 1 located between TM3, TM5, TM6, and TM7 was slightly narrower than the corresponding binding pocket 1 in D1R; such narrowness was presumably resulted from the relative bulkiness of the imidazole side chain H6.55 in D2R and D3R with respect to the amide side chain of N6.55 in D1R. Additionally, there was also a significant difference in the orientation between Y7.43 of D2R and D3R and W7.43 of D1 in which Y7.43 pointed toward extracellular side, while W7.43 was directed toward cytoplasmic side. As a result of such difference, the binding pocket 2 of both D2R and D3R were separated from pocket 1, whereas in D1R, binding pocket 2 was merged with pocket 1. Previous efforts to rationalize the structural basis of selectivity have also focused on binding regions that are not conserved among DR subfamily, with primary attention being given to ECL2.11,45,46 In D1R, C186, D187, and S188 of ECL2 were found to be in contact with l￾SPD with their counterparts in D2R and D3R being C182, I183, I184 and C181, S182, I183, respectively. Functionally relevant interactions between l-SPD and D1R, D2R, and D3R were then investigated to understand the agonistic and antagonistic mechanism of l-SPD. Hydrogen bonding, electrostatic (red dashed lines), and hydrophobic interactions were observed as major contributions to l-SPD binding. As a common binding feature, l-SPD employed its protonated nitrogen atom to form electrostatic attraction with negatively charged D3.32 in D1R, D2R, and D3R (Figure 5a− c), such a common interaction was in well agreement with the fact that D3.32 plays an essential role in ligand binding to aminergic GPCRs.45 Despite the common binding features, differences in the binding interactions of l-SPD with all three receptors were remarkable. In the l-SPD−D1R complex, the binding orientation of l-SPD with D1R was reversed as compared to the binding orientations of l-SPD with D2R and D3R (Figure 5h). Given that the structure of l-SPD has one substructure of dopamine resided on both ends, this reversion of orientation was reasonable (Scheme 1). In addition, this adjustment of binding orientation of l-SPD with D1R was optimal for the formation of two hydrogen bonds (D ring hydroxyl group D− OH with side chain of N6.55 and A ring hydroxyl group A−OH with D175 of ECL2) (Figure 5a). These two hydrogen-bonding interactions were critical for the binding affinity of l-SPD with D1R as experimental results showed that simultaneous substitution of A−OH and D−OH by methoxyl groups resulted in a ca. 60-fold loss of binding affinity,18 while monosubstitution of either A−OH or D−OH by a methoxyl group resulted in a ∼6-fold loss of binding affinity (Table S3, entries 1 and 4).18 This binding mode is further supported by site-directed mutagenesis in β2AR in which N6.55 was demonstrated to be involved in a hydrogen bonding to the β- OH group of adrenaline.47−49 Furthermore, Manivet and co￾workers showed that N6.55 in the 5-HT2B receptor is involved in direct or indirect 5-HT binding.50 In l-SPD-D2R and l-SPD-D3R complexes, the overall interaction landscape is similar to each other but with the orientation of l-SPD reversed relative to that in D1R as previously mentioned. In both l-SPD-D2R and l-SPD-D3R, the Table 1. Predicted and Experimental Binding Energies of l￾SPD with D1R, D2R, and D3R receptor predicted binding energy (kcal/mol) experimental binding energy (kcal/mol) D1R −11.00 −10.80 D2R −9.98 −9.60 D3R −10.72 −10.66 Figure 4. Correlation between predicted binding energy and experimental binding energy. The Journal of Physical Chemistry B Article 8125 dx.doi.org/10.1021/jp3049235 | J. Phys. Chem. B 2012, 116, 8121−8130