3.1 Stability of the simulated complexes
Different geometrical parameters, such as the area per lipid, root mean squared deviation (RMSD), and radius of gyration (Rg), were evaluated before structural and energetic analyses. The area per lipid analysis showed that wild-type and mutant DRD2-risperidone systems exhibited higher area per lipid values at the commencement of the simulations, decreasing to a converged behavior in the time scale of 0.1 to 0.2 µs, but reaching constant values after 0.8 µs with area per lipid values of approximately 64.0 to 67.0 Å2 for free and bound systems (Fig. S1A), in agreement with other protein-POPC-membrane systems.31 Fig. S1B shows that wild-type and mutant DRD2-risperidone systems reached a first equilibrium between 0.1 and 0.2 µs, with a final equilibrium observed after 0.8 µs, with RMSD values between 3.5 and 8.0 Å. Fig. S1C shows that similar to that observed for RMSD, all the systems achieved a first equilibrium between 0.1 and 0.2 µs, with a final convergence observed after 0.8 µs with Rg values between 28.5 and 30 Å. Based on these values, only the last 0.2 µs were employed for further analysis.
3.2. RMSF analysis of the simulated systems
RMSF analysis over the equilibrated simulation time showed that the DRD2L94A-risperidone complex exhibited reduced mobility in the second part (residues 311–365) of the cytoplasmic region between TMV and TMVI (cytoplasmic TMV-VI loop) and the intracellular TMIII-IV loop (residues 138–147) compared with DRD2WT-risperidone (Fig. S2A). The DRD2W100L-risperidone complex exhibited reduced mobility at the first (residues 257–268) and second parts (residues 311–365) of the cytoplasmic TMV-VI loop compared with DRD2WT-risperidone (Fig. S2B), except for a small fraction (residues 347–353) of the cytoplasmic TMV-VI loop, which showed an increased mobility for W100L compared to wild-type DRD2. DRD2W100A-risperidone displays increased mobility at TMIV (residues 152–174), EL2 (residues 175–193), and two regions in the cytoplasmic TMV-VI loop (residues 227–274 and 339–351) compared with DRD2WT-risperidone (Fig. S2C). In contrast, reduced mobility was observed for the TMIII-IV loop (residues 136–145) and the cytoplasmic TMV-VI loop (residues 319–333) with respect to DDR2WT-risperidone.
3.3 Protein‒ligand interactions
In the cocrystallized DRD2-risperidone complex (PDB entry 6CM4), the benzisoxasole group of risperidone interacts with seven hydrophobic and two polar residues at TMIII (F110, D114, V115, C118, T119 and I122), TMV (S193, S197 and F198), and TMVI (F382, W386 and F389). The tetrahydropyridopyrimidinone moiety of risperidone interacts with residues EL1 (W100), TMVI (F390), and TMVII (Y408, T412 and Y416) (Fig. S3).
Clustering analysis over the equilibrated simulation time provided the representative conformation for wild-type and mutant DRD2-risperidone systems. Structural analysis of the most populated conformers showed differences at the entrance to the binding pocket (Fig. 1). A more open entrance was observed for DRD2W100L-risperidone (Fig. 1C) and DRD2W100A-risperidone (Fig. 1D) than for DRD2WT-risperidone (Fig. 1A) and DRD2W100A-risperidone (Fig. 1B).
In the DRD2WT-risperidone complex, MD simulations showed that the benzisoxasole group interacted with seven hydrophobic and two polar interactions with TMIII (V115, C118, T119, and I122), EL2 (F189, V190, and S193), and TMV (S197 and F198). The tetrahydropyridopyrimidinone portion of risperidone formed interactions with EL1 (W100), EL2 (I184), the TMVI-VII loop (P405), and TMVII (Y408, S409, and T412) (Fig. 2A). Comparison with the cocrystallized DRD2-risperidone complex11 shows that a high number of residues were preserved in both systems: EL1 (W100), TMIII (V115, C118, T119, and I122), TM V (S193, S197, and F198), and TM VII (Y408, and T412).
In the DDR2L94A-risperidone complex, the benzisoxasole group was bounded by five hydrophobic and one polar contact with TMII (V83), TMIII (V115, M117, C118, and S121), and TMV (F198). The tetrahydropyridopyrimidinone portion of risperidone interacted with six hydrophobic and two polar residues at EL1 (W100 and V111), EL2 (I184 and F189), the TMVI-VII loop (P405), and TMVII (Y408, S409, and T412). Of these residues, T412 formed one hydrogen bond with the tetrahydropyridopyrimidinone portion (Fig. 2B). Comparison between the wild type and the L94A mutant showed that the benzisoxasole group was bound by residues of TMIII, EL2, and TMV in DDR2WT-risperidone, whereas residues at TMII, TMIII, and TMV participated in the stabilization of the benzisoxasole group in DDR2L94A-risperidone. The tetrahydropyridopyrimidinone portion was bound by residues at EL1, EL2, the TMVI-VII loop, and TMVII in DDR2WT-risperidone, while EL1, EL2, the TMVI-VII loop and TMVII were bound in DDR2L94A-risperidone.
In the DDR2W100L-risperidone complex, the benzisoxasole group of risperidone was bounded by eight hydrophobic residues and one neutral residue: TMIII (C118), TMV (F198), TMVI (F382, C385, W386, and F389), and TMVII (F411, L414, and G415). The tetrahydropyridopyrimidinone portion of risperidone formed interactions with EL1 (L100), TMIII (F110, and V111), EL2 (I184, and F189) and TMVII (T412) (Fig. 2C). Analysis of residues forming interactions in WT and W100L with the benzisoxasole group showed that only residues at TMIII and TMV were maintained in both systems. However, only residues EL1 (L100), EL2 (I184) and TM7 (T412) were present in the binding of the tetrahydropyridopyrimidinone portion.
In the DDR2W100A-risperidone complex, the benzisoxasole group of risperidone interacted with seven residues: TMII (V83), TMIII (M117, C118, I121, and I122), TMV (F198), and TMVII (Y416). The tetrahydropyridopyrimidinone portion of risperidone formed interactions with TMII (V91), TMIII (F110, and V111), EL2 (I184) and TMVII (T412) (Fig. 2D). Comparison with the wild type shows that only residues at TMIII (C118 and I122) and TMV (F198) were present in the binding of the benzisoxasole group in the wild type and the W100A mutant. For the tetrahydropyridopyrimidinone portion, only one residue at EL2 (I184) and TMVII (T412) was present in the coupling of this portion in the wild type and the W100A mutant.
3.3 Binding free energy calculations
Binding free energy analysis using the MMGBSA approach allowed us to determine the binding free energy (ΔGbind) value for each DRD2-ligand complex. Table 1 shows that all the complexes were energetically favorable, guided through nonpolar interactions (ΔEvdw + ΔGnpol,sol), whereas polar interactions (ΔEele + ΔGele,sol) opposed binding.
Table 1
Binding free energy components for protein‒ligand interactions of wild-type and mutated DDR2 and risperidone calculated using the MMGBSA approach (values kcal/mol)
System | ΔEvdw | ΔEele | ΔGele,sol | ΔGnpol,sol | ΔEnonpolar | ΔEpolar | ΔGbind | ΔGexp |
DDR2-risperidone | -25.30 ± 1.6 | 36.94 ± 3.7 | -33.79 ± 3.6 | -4.17 ± 0.1 | -29.47 | 3.15 | -26.32 ± 1.5 | -4.65 |
DDR2L94A-risperidone | -24.21 ± 1.5 | 26.31 ± 3.6 | -24.92 ± 3.0 | -4.38 ± 0.1 | -28.59 | 1.39 | -27.2 ± 1.4 | -6.40 |
DDR2W100A−risperidone | -22.02 ± 1.6 | 35.03 ± 8.3 | -29.76 ± 6.2 | -3.99 ± 0.2 | -26.01 | 5.27 | -20.74 ± 2.5 | -3.80 |
DDR2W100L−risperidone | -18.38 ± 2.5 | 26.93 ± 4.3 | -26.18 ± 3.8 | -3.63 ± 0.3 | -22.01 | 0.75 | -21.26 ± 2.2 | -3.84 |
Polar (ΔEele + ΔGele,sol) and nonpolar (ΔEvwd + ΔGnpol,sol) contributions. All the energies are averaged over 2000 snapshots and are in kcal/mol (standard deviation of the mean). *Values taken from Wan et al., 2018. |
The ΔGmmgbsa values were thermodynamically more favorable for DRD2WT-risperidone and DRD2L94A-risperidone than for DRD2W100L-risperidone and DRD2W100A-risperidone, implying a higher affinity of risperidone for DRD2WT and DRD2L94A, in line with the experimental ΔG value tendency (Table 1). In fact, a more negative ΔGbind value of risperidone by DRD2L94A was observed with respect to DRD2WT, in line with the experimental tendency;11 however, it is clear that another structural component may be involved in the differences in residence time at the binding pocket of the wild-type and L94A DRD2 systems.
3.4 Dissection of the per-residue free energy for DRD2-ligand complexes
Table 2 displays the per-residue energy for each residue participating in the protein‒ligand interactions of DRD2-ligand systems. In the DRD2WT-risperidone, the residues responsible for the ΔGbind value were W100, V115, C118, T119, I122, I184, F189, V190, S193, S197, F198, P405, Y408, S409 and T412. Most of these residues established nonpolar interactions (Fig. 2A).
Table 2
Per-residue free energy values for risperidone coupled to DRD2WT, DRD2L94A, DRD2W100A and DRD2W100L (values kcal/mol).
Residue | DRD2WT | DRD2L94A | DRD2W100L | DRD2W100A |
V83 | | -0.67 | | -0.456 |
V91 | | | | -0.464 |
W100 | -0.564 | -1.202 | -0.383 | |
F110 | | | -0.587 | -1.055 |
V111 | | -0.458 | -0.531 | -0.523 |
V115 | -1.774 | -0.596 | | |
M117 | | -0.592 | | -0.498 |
C118 | -2.281 | -2.21 | -0.941 | -1.781 |
T119 | -0.879 | | | |
S121 | | -0.417 | | -0.476 |
I122 | -0.521 | | | -0.614 |
I184 | -1.86 | -1.264 | -1.486 | -1.354 |
F189 | -0.962 | -0.549 | -0.582 | |
V190 | -0.839 | | | |
S193 | -0.478 | | | |
S197 | -0.991 | | | |
F198 | -1.894 | -0.603 | -0.48 | -0.445 |
F382 | | | -0.704 | |
C385 | | | -0.436 | |
W386 | | | -1.827 | |
F389 | | | -1.249 | |
P405 | -0.828 | -0.792 | | |
Y408 | -3.041 | -1.939 | | |
S409 | -0.47 | -0.531 | | |
F411 | | | -0.451 | |
T412 | -0.983 | -2.358 | -1.078 | -0.787 |
L414 | | | -0.768 | |
G415 | | | -0.97 | |
Y416 | | | | -1.359 |
In the DRD2L94A-risperidone complex, V83, W100, V111, V115, M117, C118, S121, I184, F189, F198, P405, Y408, S409, and T412 were the residues contributing to the affinity for risperidone. Of these residues, most of them established hydrophobic interactions, except T412, which formed one hydrogen bond with the tetrahydropyridopyrimidinone portion of risperidone (Fig. 2B).
In the DRD2W100L-risperidone system, the L00, F110, V111, C118, I184, F189, F198, F382, C385, W386, F389, F411, T412, L414 and G415 residues contributed to complex stabilization through nonpolar contacts (Fig. 2C). In the case of the DRD2W100A-risperidone system, the V83, V91, F110, V111, M117, C118, S121, I122, I184, F198, T412 and Y416 residues guided the affinity of the complex (Fig. 2D). This analysis shows that the affinity in wild-type and L94A was guided by a higher number of similar residues (W100, V115, C118, I184, F189, F198, P405, Y408, S409 and T412) than those observed between wild type and W100L (L100, C118, I184, F189, F198, and T412) or W100A (C118, I184, F198 and T412).
Comparison of the participation of interactions between risperidone and the residue at position 100 indicated that the tetrahydropyridopyrimidinone portion formed stronger interactions with the L94A mutant than with the wild type (Table 2), which may be responsible for the decrease in the entrance of the binding pocket cavity of L94A compared to the wild type (Fig. 1A and 1B). In contrast, W100L or W100A have an important impact on the stabilization of risperidone, which was more apparent for W100A, which even lost interactions with the tetrahydropyridopyrimidinone portion (Table 2 and Fig. 2D). These differences may be linked with the increases in the sizes of the cavity entrances for the DRD2W100L-risperidone (Fig. 1C) and DRD2W100A-risperidone systems (Fig. 1D) compared to the DRD2WT-risperidone (Fig. 1A) and DRD2L94A-risperidone systems (Fig. 1B).
3.5 Principal component analysis
PC analysis identified the main motions along the wild-type and mutant DRD2-ligand systems. The first two eigenvectors include the major eigenvalues; these contained 54.7, 45.4, 57.4, and 54.4% of the total flexibility of the DRD2WT-risperidone, DRD2L94A-risperidone, DRD2W100L-risperidone and DRD2W100A-risperidone systems, respectively. Projection over the phase space of the first (PC1) and second (PC2) eigenvectors shows that the DRD2W100A-risperidone system (Fig. 3D) covers a more extensive distribution in the essential subspace than the DRD2WT-risperidone systems (Fig. 3A), indicating a larger conformational entropy for ligand recognition for W100A with respect to wild-type DRD2. The DRD2W100L-risperidone (Fig. 3C) and DRD2WT-risperidone systems exhibit similar distributions along the essential subspace, indicating similar conformational behavior in risperidone recognition. However, DRD2L94A-risperidone systems indicate a more compact distribution in the essential subspace with respect to the wild type (Fig. 3B), suggesting an unfavorable entropy contribution upon ligand recognition compared with the other systems.
The diagonalized covariance matrix over the backbone atoms provides the following values: DRD2WT-risperidone (53.7 nm2), DRD2L94A-risperidone (26.3 nm2), DRD2W100L-risperidone (50.6 nm2), and DRD2W100A-risperidone (81.2 nm2). These results suggest that the binding of risperidone to DRD2L94A reduces the number of conformational states regarding DRD2WT. The molecular recognition of risperidone on DRD2WT and DRD2W100L exhibits similar numbers of conformers. In contrast, the binding of risperidone to DRD2W100A increases the number of conformational states in solution compared to DRD2WT. Correlation of these covariance values with RMSF analysis indicated that the major contributor to the differences in mobility between the wild type and the mutants was the cytoplasmic TMV-VI loop for DRD2WT-risperidone, DDR2L94A-risperidone, and DDR2W100L-risperidone (Fig. S2). For DDR2W100A-risperidone and DDR2WT-risperidone, the main contributors were the cytoplasmic TMV-VI loop, TMIV, and EL2.
3.6 Energy minimization of wild-type and mutant DRD2-risperidone complexes
The YASARA minimization server was used to minimize the most populated wild-type and mutant DRD2-risperidone complexes obtained during the equilibrated simulation time (i.e., the last 0.2 µs) through clustering analysis.30 Energy minimization findings revealed increases in the free energy for DRD2L94A-risperidone and DRD2W100L-risperidone with respect to DRD2WT-risperidone, whereas a decrease was observed for DRD2W100A-risperidone compared with DRD2WT-risperidone (Table 3). These results indicate that L94A and W100L optimize the structure, whereas W100A destabilizes the structure with respect to the wild type.
Table 3
RMSD (Å) values and total free energy after energy minimization of wild-type and mutant DRD2.
Systems | Energy after minimization (kj/mol) | RMSD (Å) |
DDR2WT-risperidone | -235938.4 | |
DDR2L94A-risperidone | -234063.4 | 2.18 |
DDR2W100L-risperidone | -236213.3 | 3.70 |
DDR2W100A-risperidone | -232787.4 | 3.50 |
These differences correlate with the significantly lower mobility of DRD2L94A-risperidone and the increased dynamic motion of DRD2W100A-risperidone compared to DRD2WT-risperidone, but not much with the slightly lower mobility DRD2W100L-risperidone compared to DRD2WT-risperidone. Analysis of RMSD between the four conformers showed that L94A less drastically impacted the structure of the protein compared with wild-type DDR2. In contrast, W100L and W100A suggest a critical change in the DRD2 structure compared to wild-type DDR2.
3.7 Structural analysis of W100 mutations in wild-type and mutant DRD2-risperidone complexes
Analysis of the most populated complexes showed that the side chain of W100 in DRD2WT-risperidone is stabilized by contacts of I184, L94 and P405, similar to the crystallographic structure (Fig. S4A). The same interactions were also observed for the side chain of W100 in DRD2L94A-risperidone, together with the addition of interactions of the side chain with G98 (Fig. S4B). In contrast, the side chain of L100 in DRD2W100L-risperidone only interacts with L94 (Fig. S4C), and the side chain of A100 in DRD2W100A-risperidone only forms interactions with C107 (Fig. S4D).