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