Supplementary Material
Journal: Amino Acids
Title: Insilico Study of the A2AR-D2R Kinetics and Interfacial Contact Surface for Heteromerization
Amresh Prakash and Pratibha Mehta Luthra*
Medicinal Chemistry Division, Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, North Campus, Mall Road, Delhi 110007, India
*Corresponding author. Tel.: +91 11 27662778; fax: +91 11 27666248.
E-mail addresses: ,
Supplementary Material Figures
Figure S1. Sequence alignment of D2R with selected templates: D3R, β2R and A2AR using Clustal-X (2.0.11). Conserve sequences were represented as symbol (*) and semiconserve residues were denoted with symbol (:). Red box represent the conserved "DRY" motif on all four GPCR protein.
Figure S2. Ramachandran ((RC) plot of the D2R and A2AR model. The distribution of the D2R and A2AR residues (black dots) were shown in color: most favorable (red), additional allowed (yellow), and generously allowed (light yellow) regions. The 98.6 % and 98.9% residues of D2R and A2AR respectively were in allowed region of RC-plot.
Figure S3a. The MD simulation of 10 ps provided to optimized the D2R , A2AR and D2R -A2AR complex. The graph represented the frame dependent displacement of molecule and the trajectory touching the x-axis of graph represented the best optimized state of molecule.
Figure S3b. The MD simulation of 10 ps provided to optimized the D2R , A2AR and D2R -A2AR complex. The lower three graph represented the change in energy with respect to time for receptor D2R , A2AR and D2R-A2AR. The trajectory energy corresponding to frame showed the lowest energy of molecule.
A2AR H-bond graph and D2R H-bond graph
Figure S4 HB Plot representation. Helices can be identified as strips directly adjacent to the diagonal, antiparallel beta strands by strips perpendicular to the diagonal, and parallel beta strands by off-diagonal strips parallel to the diagonal. A2AR residues were represented as blue circle, D2R residues were represented as pink circle, and A2AR-D2R interaction residues represented by green circles. Circle 1 and 2 showed the N-terminus (A2AR) interaction with C-terminus (D2R) and circle 7 represented the perturbations at C-terminus of both receptor where as circle 3 represented the tertiary interaction between the Pro61 of A2AR with Lys149 of D2R. Tertiary interactions represented here is symmetric to diagonal.Three classes of hydrogen bondings are distinguished by color coding; namely, short (distance smaller than 2.5Å between donor and acceptor) red, intermediate (between 2.5 and 3.2 Å) tv-red and long hydrogen bonds (greater than 3.2 Å) pink.
Figure S5. All atom superimposed view of Crystal structure of A2AR; PDB ID: 3EML (color green) and Modeled A2AR (PMDB: PM0077535) with loops (color cyan) at PyMol showed rmsd value 0.252. To validate the modeled structure docking simulation method was implied with know A2AR antagonists. These antagonists set consisted with co-crystallized pose of ZM241385 (color by CPK). The modeled structure of A2AR with
Figure S6. Docked pose of A2AR antagonist ZM241385 with A2AR (PMDB: PM0077535). Cartoon view of A2AR and docked pose of A2AR antagonists (ZM241385) on PyMol. Co-crystallized pose of ZM241385 docked with modeled A2AR. Transmembrane (TM) and Extra cellular loops (ECLs) are color by rainbow and antagonists colored by CPK. Yellow dotted line showed polar interaction with E169 and N253.
Figure S7. Cartoon view of D2R in PyMol. Dopamine docked pose with D2R showed polar interaction with V91, E95, S409 and Y416 (polar interaction shown by yellow dotted line). W413 and Y416 showed aromatic stacking and hydrophobic interaction with catechol ring and amino tail engaged in polar interaction with D114 (most conserved residue of D2-like receptors active site) which is not clearly visible in the present pose. the site directed mutagenesis study data, showed that binding site is expected to be lined by highly conversed residues among which D114 (TM3), S193, 194, 197 (TM5), Y416 (TM7) and F389 (TM6) and are of crucial importance ( Ehrlich et al. 2009).
Supplementary Material Table
Interfacial geometric properties calculated using ProtorP summarized in table 1.
Table S1. A2AR and D2R Interface Parameter Value
Interface Residue Segments 7
Interface Accessible Surface Area (Å2) 469.23
Atoms in Interface 143
% Polar Atoms Contribution to Interface 45.02
% Non-Polar Atoms Contribution to Interface 35.06
% Neutral Atoms Contribution to Interface 18.96
Residues in Interface 15
% Polar Residues in Interface 60.00
% Non-Polar Residues in Interface 26.67
% Charged Residues in Interface 13.33
Residues on Surface 109
% Polar Residues on Surface 32.11
% Non-Polar Residues on Surface 42.20
% Charged Residues on Surface 25.69
Planarity (Å) 0.982
Eccentricity 0.857
Secondary Structure in Interface Beta
Secondary Structure in Interface Helix
% Alpha Character in Interface 20.00
% Beta Character in Interface 17.00
Secondary Structure on Surface Beta
Secondary Structure in Interface Helix
% Alpha Character on Surface 40.69
% Beta Character on Surface 30.37
Hydrogen Bonds 8
Salt Bridges 3
Disulphide Bonds 2
Bridging Water Molecules 0
Gap Volume (Å3) 2392.88
Gap Volume Index (Å) 2.55
*Software did not consider loops and bends
Table S2. Per Domain Analysis of the Propension to Disorder and to Aggregation of A2AR and D2R, Receptor Proteins (In Brackets the Code Identifying them in the Swiss-Prot Database).
A2A R (P27274) / D2R (P14416) / A2AR (P27274) / D2R (P14416)
N-term / 0.386 / 0.394 / 0.455 / 0.040
TM1 / 0.000 / 0.000 / 0.637 / 0.741
ICL1 / 0.023 / 0.009 / 0.457 / 0.229
TM2 / 0.117 / 0.092 / 0.525 / 0.579
ECL1 / 0.100 / 0.100 / 0.612 / 0.443
TM3 / 0.013 / 0.054 / 0.612 / 0.430
ICL2 / 0.055 / 0.119 / 0.380 / 0.288
TM4 / 0.022 / 0.004 / 0.489 / 0.559
ECL2 / 0.163 / 0.092 / 0.080 / 0.000
TM5 / 0.000 / 0.042 / 0.773 / 0.801
ICL3 / 0.272 / 0.620 / 0.199 / 0.074
TM6 / 0.000 / 0.012 / 0.625 / 0.781
ECL3 / 0.012 / 0.050 / 0.317 / 0.123
TM7 / 0.037 / 0.067 / 0.508 / 0.459
C-Term / 0.613 / 0.029 / 0.034 / 0.336
TM=Transmembrane; ICL=intracellular loop; ECL=extracellular loop
Values of DI and AI greater than 0.5 are highlighted in bold. The highest observed value of the parameters appears in bold underlined.
Table S3. Disulphide bridges and cation–π interaction of A2AR, D2R and A2AR-D2R complex Disulphide bridges: Between sulphur atoms of cysteines within 2.2 Å
Position Residue PositionResidue Distance
A2AR
259 CYS 262 CYS 2.04
71 CYS 159 CYS 2.03
74 CYS 146 CYS 2.10
77 CYS 166 CYS 2.03
D2R
107 CYS 182 CYS 2.04
cation–π Interactions within 6 Å
Position ResiduePosition ResidueD(cation-Pi)Angle
A2AR
43 TYR 122 LYS 5.61 116.82
103 TYR 199 ARG 5.72 84.16
D2R
263 PHE 275 ARG 4.70 74.24
429 PHE 370 LYS 6.00 100.00
Table S4. The long cytoplasmic loop (ICL3) of D2R containing 160 amino acids residues connecting TM5 and TM6 where as A2AR longest cytoplasmic loop (ICL3) containing 122 amino acid residues after TM7. Secondary structure feature of A2AR (ICL3) and D2R (ICL3).
A2AR (ICL3) D2R (ICL3)
LYS315 ALA321 S ALA234 PRO239 S
LEU324 GLN333 S LEU240 GLY242 L
SER335 TYR340 S ASN243 THR245 S
HIS341 PRO354 L HIS246 LEU252 L
GLU355 TYR361 S CYS253 SER262 H
ALA362 SER374 L PHE263 ARG268 L
GLN375 HIS388 H ARG269 GLU271 S
GLU389 CYS394 L ALA273 GLU280 H
PRO395 GLY399 S MET281 SER286 L
LEU400 ASP401 L THR287 ARG294 H
ASP402 VAL411 H TYR295 PRO297 L
ILE298 ASP309 H
PRO310 HIS312 L
HIS313 HIS316 H
SER317 PRO319 L
ASP320 GLU326 H
LYS327 GLY329 L
HIS330 ILE337 H
ALA338 ILE340 L
PHE341 GLN344 H
THR345 PRO347 L
ASN348 LYS350 H
THR351 SER359 L
ARG360 ARG361 H
LYS362 GLN366 L
LYS367 HIS398 H
CYS399 PRO404 L
PRO405 THR428 H
PHE429 ILE431 L
GLU432 LEU441 H
S = Sheet; H = Helix; L = Loop