Cell Reports, Volume 29
Supplemental Information
Molecular Mechanism for Ligand Recognition
and Subtype Selectivity of a2C Adrenergic Receptor
Xiaoyu Chen, Yueming Xu, Lu Qu, Lijie Wu, Gye Won Han, Yu Guo, Yiran Wu, QingtongZhou, Qianqian Sun, Cenfeng Chu, Jie Yang, Liu Yang, Quan Wang, Shuguang Yuan, LingWang, Tao Hu, Houchao Tao, Yaping Sun, Yunpeng Song, Liaoyuan Hu, Zhi-JieLiu, Raymond C. Stevens, Suwen Zhao, Dong Wu, and Guisheng Zhong
Figure S1. Engineered construct, biochemical analysis, crystals of α2CAR, and
binding affinity of RS79948 on α2CAR. Related to Figure 1. (A) Schematic diagram
of the engineered α2CAR construct. To stabilize α2CAR in vitro, we truncated 28 amino
acids at the N-terminus and 4 amino acids at the C-terminus and replaced the ICL3 loop
(242-371) with PGS (in blue). Three thermal-stabilized mutations, V1223.23W,
I1393.40A, and F3916.44W (in red) were integrated into this construct. Disulfide bonds
are displayed as dashed lines between cysteine residues 124 and 202, and 408 and 412,
which are shown in orange. (B) Analytical size-exclusion chromatography (aSEC). No
Mut: construct with N/C-terminal truncated, ICL3 replaced by PGS and without
mutation; Mut: the engineered construct with V1223.23W, I1393.40A, and F3916.44W. (C)
CPM thermostability ramping assay of α2CAR. The three mutations increase TM value
by about 10 degrees (No Mut: 46.07℃; Mut: 56.45℃). (D) Crystal images of α2CAR
bound with RS79948. White scale bar: 90 μm. (E) Saturation binding assay for [3H]-
Rauwolscine specific binding on WT and crystallized α2CAR. Kd values are presented
(data are mean ± SD; n = 3.). (F) Competition binding curves of RS79948. Ki values
are presented (data are mean ± SD; n = 3.). Experiments were performed in technical
triplicate and data are shown as mean ± s.e.m.
Figure S2. The conformational comparison of TM6 between α2CAR and the active-
(PDB ID: 3SN6) and inactive-state β2AR (PDB ID: 2RH1) suggests that the α2CAR
structure is in an inactive state. Related to Figure 1.
Figure S3. Electron density maps of the ligand RS79948 and its interacting
residues in the ligand binding pocket of α2CAR-RS79948 structure. Related to
Figure 2. (A) The |Fo|-|Fc| polder omit map (green mesh) is contoured at 3σ. (B) The
2|Fo|-|Fc| omit map (blue mesh) is contoured at 1.0σ. RS79948 (yellow). Key residues
in the binding pocket (cyan).
Figure S4. Pocket comparison of α2CAR structure with other aminergic GPCRs.
Related to Figure 3. (A) Vertically-sliced surface representation of the receptors with
their ligands. (B) Ligands are shown as surfaces with their minimum and maximum
distance to the outside of the lipid bilayer (black line) given. The top and bottom of the
α2CAR ligand, RS79948, is marked as dotted lines. (C) Histogram of the ligands’
minimum (red) and maximum (cyan) distance to extracellular space. α2AAR, α2CAR,
D2R (6CM4), D4R (5WIU), D3R (3PBL), 5-HT2CR (6BQH), 5-HT2BR (5TVN), 5-
HT1BR (5V54), β1AR (4AMJ), β2AR (2RH1), H1R (3RZE), M1R (5CXV), M3R
(4DAJ), M2R (3UON), M4R (5DSG). (D) Histogram of pocket volume across the
aminergic receptors.
Figure S5. The loosened helix at the top of TM4 in α2CAR is essential for receptor
activation by endogenous nonselective agonists. Related to Figure 3. (A)
Superposition of α2CAR and β2AR (PDB: 2RH1) shows that α2CAR has a much shorter
TM4. (B) The sequence interrupting the α-helix structure in α2CAR compared with
β2AR. (C) P185L mutation on α2CAR attenuated the potency of endogenous agonists
norepinephrine and epinephrine. (D) P185L mutation on α2CAR had no or slight effect
on α2-agonists clonidine and dexmedetomidine. Data are shown as mean ± SEM with
two independent experiments performed in triplicate.
Figure S6. Adrenergic receptors share highly conserved pocket residues for
epinephrine binding. Related to Figure 3. (A) Alignment of 12 defined aminergic
conserved orthosteric binding sites among adrenergic receptors. (B) Comparison of
polar binding residues of β2AR-epinephrine (PDB: 4LDO, cyan) and docking α2CAR-
epinephrine (wheat).
Figure S7. Similarity of α2CAR and α2AAR binding pockets bound with RS79948
or OPC-28326. Related to Figure 4. (A) Top of α2CAR and α2AAR binding pockets.
Residues of α2CAR and α2AAR are shown as brown and green sticks, respectively. (B)
OPC-28326 (in pink) interacts with similar pocket residues as α2CAR (sticks shown in
wheat) and α2AAR (sticks shown in green) in the docking models.
Figure S8. Molecular docking of JP1302 in the orthosteric pocket of α2CAR.
Related to Figure 4. (A) Competition binding of JP1302 on the WT and crystallized
α2CAR. Ki values are shown (data are mean ± SD; n = 3.). (B) JP1302 competitively
antagonizes the UK 14,304-induced inhibition of forskolin-stimulated cAMP
accumulation in CHO cells transiently transfected with α2CAR. Data are shown as mean
± SEM with two independent experiments performed in triplicate. (C) TC1515 is an
analogue of JP1302 with 1-methylpiperazine changed to morpholine. (D) TC1515 had
no antagonism on UK 14,304-inducecd cAMP assay inhibition. Data are shown as
mean ± SEM with three independent experiments performed in triplicate. (E) Key
residues involved in JP1302 binding are shown as green and wheat sticks. The
conserved site in aminergic pocket D3.32 is shown as green sticks and the salt bridge
between α2CAR and JP1302 is displayed as green dashed lines. (F) Schematic
representation of the interactions between α2CAR and JP1302. The salt bridge is shown
as green dashed line, and π–π and cation-π interactions are shown as red dashed lines.
Table S1. Data Collection and Structure Refinement Statistics. Related to Figure
2.
α2CAR-RS
(PDB: 6KUW )
Data Collection
Space group P212121
Cell dimensions
a, b, c (Å) 74.483, 78.737, 190.445
α,β,γ (o) 90.0, 90.0, 90.0
Resolution (Å) 47.04-2.80 (2.87-2.80)a
Rmerge (%) 11.0 (72.1)
I/σI 8.15 (1.74)
Completeness (%) 94.4 (82.1)
CC1/2 99.6 (60.4)
Redundancy 3.75 (3.23)
Refinement
Resolution (Å) 47.04-2.80
No. reflections 26,776
Rwork/Rfree (%) 23.0/26.2
No. atoms
Protein
7,370
Ligand 50
Lipids and waters 197
B-factors (Å2)
α2CAR
A B
65.0 84.0
PGS 120.9 116.6
Ligand 69.5 71.4
Lipids and waters 77.4 87.5
R.M.S. deviations
Bond lengths (Å) 0.008
Bond angles (o) 0.98 aValues in parentheses are for highest-resolution shell.
Table S2. Analyses of mutation effects by cAMP assay. Related to Figure 2,
Figure4, Figure S6.
UK14,304 RS79948 JP1302 OPC-28326
α2C WT 7.89±0.07 8.70±0.16 5.58±0.10 7.02±0.11
L128A 8.17±0.30 6.95±0.14*** 5.84±0.16 -
L128W 8.05±0.09 6.30±0.16*** 6.22±0.09* -
L204A <5.00 - - -
L204I 7.81±0.54 7.50±0.17*** 5.26±0.10 -
W395A <5.00 - - -
F398A 7.63±0.25 9.13±0.14 6.21±0.28 -
F419A 7.07±0.07* - - -
F419W 7.63±0.18 8.71±0.09 6.64±0.04*** -
F423A <5.00 - - -
F423W 6.45±0.21*** - - -
Y427A <5.00 - - -
D206A 7.50±0.08 8.99±0.15 5.36±0.15 7.08±0.27
R409A 8.01±0.22 8.88±0.09 5.63±0.29 7.00±0.34
Y405T 7.75±0.16 8.93±0.12 5.24±0.12* 7.17±0.15
D206A/R409A/Y405T 7.37±0.90 9.17±0.11 <5.00 <5.00
Analysis of pEC50 values in cAMP assays. Data are mean pEC50 ± s.e.m. from fitting
concentration-response data to nonlinear regression (3 parameter) analysis; n ≥ 3
independent experiments performed in duplicate for all mutants and wild type. The
pEC50 values of mutations were compared with that of wild type by two-way ANOVA:
* P < 0.05, * * P < 0.01, * * * P < 0.001. Statistical analyses were not performed on
some mutations with pEC50 < 5 due to lack of response and non-convergence of
nonlinear regression analysis.
Table S3. Primer sequence for oligonucleotides used in the cAMP assays. Related
to Key Resource Table.
Oligonucleotides
Primers for cloning α2CAR-WT into pcDNA 3.1 (+) vector
α2CAR_WT_forward:TACAAGGACGATGACGATGCTGGGCGC
GCCATGGCCTCACCCGCACTTGCTGCCGCCCTT
This paper N/A
α2CAR_WT_reverse:GGTTTAAACGGGCCCTCTAGACTCGAGT
TACTGTCGGAAGCCCCTTCGCCTGCGCCTGAA
This paper N/A
Primers for site-direct mutagenesis
L128A_forward:CGGGCAGGTGTGGTGTGGAGTCTATGCAGC
ACTTGATGTGCTCTTTTGCACGT
This paper N/A
L128A_reverse:ACGTGCAAAAGAGCACATCAAGTGCTGCATA
GACTCCACACCACACCTGCCCG
This paper N/A
L128W_forward:CGGGCAGGTGTGGTGTGGAGTCTATTGGGC
ACTTGATGTGCTCTTTTGCACGT
This paper N/A
L128W_reverse:ACGTGCAAAAGAGCACATCAAGTGCCCAAT
AGACTCCACACCACACCTGCCCG
This paper N/A
L204A_forward:TGGGGCAGCCTATCCACAGTGCGGAGCAAA
TGACGAAACGTGGTACATACTTA
This paper N/A
L204A_reverse:TAAGTATGTACCACGTTTCGTCATTTGCTCCG
CACTGTGGATAGGCTGCCCCA
This paper N/A
L204I_forward:GATGGGGCAGCCTATCCACAGTGCGGAATCA
ATGACGAAACGTGGTACATACTTAGT
This paper N/A
L204I_reverse:ACTAAGTATGTACCACGTTTCGTCATTGATTC
CGCACTGTGGATAGGCTGCCCCATC
This paper N/A
W395A_forward:GGTAATGGGAGTTTTCGTGCTGTGTGCATTC
CCATTCTTCTTTTCATACTCAC
This paper N/A
W395A_reverse:GTGAGTATGAAAAGAAGAATGGGAATGCAC
ACAGCACGAAAACTCCCATTACC
This paper N/A
F398A_forward:GAGTTTTCGTGCTGTGTTGGTTCCCAGCATTC
TTTTCATACTCACTGTACGGCAT
This paper N/A
F398A_reverse:ATGCCGTACAGTGAGTATGAAAAGAATGCTG
GGAACCAACACAGCACGAAAACTC
This paper N/A
F419A_forward:GGCTTGCCAAGTCCCTGGCCCTCTGGCAAAG
TTCTTTTTCTGGATCGGCTACT
This paper N/A
F419A_reverse:AGTAGCCGATCCAGAAAAAGAACTTTGCCAG
AGGGCCAGGGACTTGGCAAGCC
This paper N/A
F419W_forward:GGCTTGCCAAGTCCCTGGCCCTCTGTGGAAG
TTCTTTTTCTGGATCGGCTACT
This paper N/A
F419W_reverse:AGTAGCCGATCCAGAAAAAGAACTTCCACA
GAGGGCCAGGGACTTGGCAAGCC
This paper N/A
F423A_forward:TCCCTGGCCCTCTGTTCAAGTTCTTTGCATGG
ATCGGCTACTGTAATAGCAGTCT
This paper N/A
F423A_reverse:AGACTGCTATTACAGTAGCCGATCCATGCAA
AGAACTTGAACAGAGGGCCAGGGA
This paper N/A
F423W_forward:CCCTGGCCCTCTGTTCAAGTTCTTTTGGTGG
ATCGGCTACTGTAATAGCAGTC
This paper N/A
F423W_reverse:GACTGCTATTACAGTAGCCGATCCACCAAAA
GAACTTGAACAGAGGGCCAGGG
This paper N/A
Y427A_forward:GTTCAAGTTCTTTTTCTGGATCGGCGCATGT
AATAGCAGTCTCAATCCGGTTA
This paper N/A
Y427A_reverse:TAACCGGATTGAGACTGCTATTACATGCGCC
GATCCAGAAAAAGAACTTGAAC
This paper N/A
D206A_forward:CAGCCTATCCACAGTGCGGACTTAATGCCG
AAACGTGGTACATACTTAGTAGCTG
This paper N/A
D206A_reverse:CAGCTACTAAGTATGTACCACGTTTCGGCAT
TAAGTCCGCACTGTGGATAGGCTG
This paper N/A
R409A_forward:TTTCATACTCACTGTACGGCATTTGCGCCGA
GGCTTGCCAAGTCCCTGGCCCTCT
This paper N/A
R409A_reverse:AGAGGGCCAGGGACTTGGCAAGCCTCGGCG
CAAATGCCGTACAGTGAGTATGAAA
This paper N/A
Y405T_forward:TTCCCATTCTTCTTTTCATACTCACTGACCGG
CATTTGCCGAGAGGCTTGCCAAG
This paper N/A
Y405T_reverse:CTTGGCAAGCCTCTCGGCAAATGCCGGTCAG
TGAGTATGAAAAGAAGAATGGGAA
This paper N/A
Y405T/R409A_forward:CCCATTCTTCTTTTCATACTCACTGAC
CGGCATTTGCGCCGAGGCTTGCCAA
This paper N/A
Y405T/R409A_reverse:TTGGCAAGCCTCGGCGCAAATGCCGG
TCAGTGAGTATGAAAAGAAGAATGGG
This paper N/A
P185L_forward:GCTCATTAGCGCTGTGATCTCATTTCTGCCCC
TGGTGAGTCTTTACCGACAAC
This paper N/A
P185L_reverse:GTTGTCGGTAAAGACTCACCAGGGGCAGAAA
TGAGATCACAGCGCTAATGAGC
This paper N/A
Primers for site-direct mutagenesis on pcDNA 3.1-α2AAR-WT
aK144A_forward:TACGCAGGCGATCGAGTACAACTTGGCAA
GGACCCCTCGCCGTATCAAAGCTA
This paper N/A
aK144A_reverse:TAGCTTTGATACGGCGAGGGGTCCTTGCCA
AGTTGTACTCGATCGCCTGCGTA
This paper N/A
aR405G_forward:CACCGCCGTGGGATGTAGCGTCCCTGGAAC
TTTGTTCAAGTTCTTCTTCTG
This paper N/A
aR405G_reverse:CAGAAGAAGAACTTGAACAAAGTTCCAGGG
ACGCTACATCCCACGGCGGTG
This paper N/A
chimera-G408R_forward:TTGCCGAGAGGCTTGCCAAGTCCCTC
GTACTTTGTTCAAGTTCTTCTTCTGGT
This paper N/A
chimera-G408R_reverse:ACCAGAAGAAGAACTTGAACAAAGT
ACGAGGGACTTGGCAAGCCTCTCGGCAA
This paper N/A
chimera_forward:CCTTTCTTCTTCACATACACGCTCTACGGCA
TTTGCCGAGAGGCTTGCCAAGTCCCTGGCACTTTGTTCAAG
TTCTTCTT
This paper N/A
chimera_reverse:CCAGAAGAAGAACTTGAACAAAGTGCCAGG
GACTTGGCAAGCCTCTCGGCAAATGCCGTAGAGCGTGTAT
GTGAAGAAGA
This paper N/A