Structure, Volume 23

Supplemental Information

The Importance of Ligand-Receptor Conformational

Pairs in Stabilization: Spotlight on the N/OFQ G
Protein-Coupled Receptor
Rebecca L. Miller, Aaron A. Thompson, Claudio Trapella, Remo Guerrini, Davide
Malfacini, Nilkanth Patel, Gye Won Han, Vadim Cherezov, Girolamo Cal, Vsevolod
Katritch, and Raymond C. Stevens

SUPPLEMENTAL DATA

Figure S1, related to Table 1: Ligand electron densities for SB-612111 and Compound-35
within the NOP binding pocket. Two complementary views of the electron densities for the cocrystallized ligands in the orthosteric binding pocket of NOP (blue mesh 2mFo-DFc map
contoured at 1). Panels (A) and (B) show the electron densities for ligands SB-612111, purple,
while panels (C) and (D) show densities for Compound-35, orange, in their respective monomers
of the crystal structure.

Figure S2, related to Figure 1: Primary data for BRET experiments. (A) Concentrationresponse curve to determine agonist Emax for N/OFQ (10 pM - 10 M), BRET data are mean
SEM of 6 experiments performed in duplicate. N/OFQ was tested in membranes taken from
HEK293 cells stably co-expressing the human NOP (RLuc tagged, NOP-RLuc) receptor and the
G1 (RGFP linked, G1-RGFP) protein subunit. The peptide concentration dependently stimulated
BRET ratio showing high values of potency 9.26 (8.60-9.92) and maximal effect (Emax) of 0.45
0.03 stimulated BRET ratio (Figure 1). This value of potency is similar to those found in stimulated
[35S]GTPS binding assay studies (e.g. pEC50 = 8.95) (Fischetti et al., 2009). Panels (B-F) show
the concentration-response curves of N/OFQ (10 pM - 10 M) in the presence of vehicle or
antagonists (B) Trap-101 100 nM, (C) J-113397 100 nM, (D) SB-612111 10 nM, (E) C-24 10 nM,
and (F) C-35 10 nM. Data are shown as E/Emax (Emax N/OFQ = 1) and are mean SEM of at least
4 separate experiments performed in duplicate.

Figure S3, related to Figure 2: Strong bi-lobed positive density observed in conserved Na+
binding pocket. Examining the difference map (|Fo-Fc| contoured at 3) of NOPSB (molecule
A), we observe a strong bi-lobed region of positive density within the transmembrane core of the
receptor flanked by residues D972.50, N1333.35, S1373.39, and N3117.45, highly conserved residues
in Class A GPCRs. Coordination of a sodium ion in this site has been crystallographically
established in several class A GPCRs, including the A2A Adenosine Receptor (A2AAR), 2Adrenergic Receptor (2AR), Protease-Activated Receptor 1 (PAR1), and closely related Opioid Receptor (-OR), and hypothesized for a majority of class A GPCRs, including NOP
(Katritch et al., 2014). Allosteric modulation of NOP binding by Na+ at physiological
concentrations has been reported in the literature, further supporting sodium binding in NOP and
its role in receptor function (Ardati et al., 1997; Butour et al., 1997). Notably, mutation of one of
these acidic residues N1333.35 to a bulky and basic tryptophan confers constitutive activity to
NOP through several G-protein coupled signaling pathways (Kam et al., 2002). Although the
lower lobe of this positive density is compatible with the Na+ positioning in other class A
structures, definitive assignment of Na+ requires identification of all water molecules in its
coordination shell, which is not possible at the current resolution.

Figure S4, related to Figure 3: Cross-docking validation and experimental docking results.
(A) C-24 docking, with the original pose in NOPC-24 crystal structure shown with green carbons,
(B) C-35 docking, with the original pose in NOPC-35 crystal structure shown with orange
carbons, (C) SB-612111 docking, with the original pose in NOPSB-612111 crystal structure
shown with magenta carbons. Panels (D), (E), and (F) show an overlay of the two binding modes
of J-113397, Trap-101, and JTC-801, respectively.

Table S1, related to Figure 1: Explicit values for NOP ligand metrics. Receptor melting temperature
(Tm, mean SEM), antagonist potency (pKB, mean CL95%), ligand molecular weight (MW in Da),
calculated ligand lipophilicity (LogD) and affinity (pKi) for NOP. Lipophilicity LogDs (the pKa- and pHdependent version of LogP) were calculated using the JChem extension for Microsoft Excel with pH=7.5,
[NaCl] = 0.5M. References given are for affinity measurements, while the superscript a indicates where
the pKi was calculated from a given experimental Ki using the equation pKi = Log10 (Ki).
Compound

Activity

Compound-24

Antagonist

Compound-35

Antagonist

SB-612111

Antagonist

Ac-RYYRWK-NH2 Partial Agonist

Agonist
MCOPPB 3HCl
J-113397
JTC-801
Ac-RYYRIK-NH2
Trap-101
UFP-112
Buprenorphine
UFP-113
SCH-221510
Ro-656570
Nociceptin
(N/OFQ)

Antagonist
Antagonist
Partial Agonist
Antagonist
Agonist
Partial Agonist
Partial Agonist
Agonist
Agonist

Antagonist
MW Lipophilicity Affinity
Potency
(Da)
(LogD)
(pKi)
(pKB)
10.40
69.9 ( 0.1)
506.5
0.91
9.62
(9.26-11.53)
9.85
68.4 ( 0.1)
517
3.96
9.14
(8.89-10.81)
9.57
62.6 ( 0.1)
459.4
5.16
9.18
(9.16-9.98)
59.1 ( 0.5)
1354.3
-8.45 9.14a
57.5 ( 0.2)
534.1
-0.37 10.07
8.36
435.7
2.50
8.56
56.0 ( 0.1)
(7.66-9.07)
55.4 ( 0.2)
456.9
3.86 8.08a
54.5 ( 0.3)
1281.2
-8.87 8.82a
8.30
54.4 ( 0.1)
434.0
2.61
8.65
(7.87-8.73)
54.2 ( 0.2)
2738
-28.89 10.55
53.7 ( 0.1)
504.1
1.70 7.11a
53.2 ( 0.1)
2724
-29.39 10.26
52.0 ( 0.2)
434.0
3.74 9.52a
51.8 ( 0.2)
456.4
2.02 9.28a
Stability
(C)

Agonist 48.7 ( 0.7)

2379

Agonist 48.5 ( 0.4)

443.5

Nor-BNI

Antagonist 48.7 ( 0.2)

770.8

UFP-101

Antagonist 47.9 ( 0.3)

NNC 63-0532

Apo
N/OFQ(1-13)-NH2

-- 47.9 ( 0.2)
Agonist 47.3 ( 0.3)

7.34
2706
(6.73-7.94)
-1952

Affinity
Reference
(Fischetti et al., 2009)
(Trapella et al., 2009)
(Spagnolo et al., 2007)
(Dooley et al., 1997)
(Hayashi et al., 2009)
(Bigoni et al., 2000)
(Shinkai et al., 2000)
(Dooley et al., 1997)
(Trapella et al., 2006)
(Arduin et al., 2007)
(Khroyan et al., 2015)
(Arduin et al., 2007)
(Varty et al., 2008)
(Zaveri, 2003)

-22.22 10.10a

(Khroyan et al., 2015)

2.03

8.14a

1.41

5.36a

(Thomsen and Hohlweg,

2000)
(Munro et al., 2013)

-29.69

10.20

(Calo et al., 2002)

--20.29

--9.21a (Charoenchai et al., 2008)

Table S2, related to Figure 3: Chemical structures of various NOP antagonists.

Table S3, related to Figure 3: Cross-docking validation and experimental docking results
for various NOP antagonists. Cross-docking validation results of each co-crystallized ligand
into each of the three NOP crystal structures. Binding scores (in kJ/mol) are given for each
ligand docked into both Rigid and Flexible models of NOP. The flexible NOP model
incorporates extensive side conformation and water molecule sampling, whereas the rigid
docking models do not. For compounds J-113397, Trap-101, and JTC-801, the binding score of
two binding modes with comparable energies (modes I & II, see Figures 3 & S4) are given.
Asterisks indicate that no significant binding score was found.
Cross-Docking Validation Results
Binding Score, kJ/mol
(RMSD, )
Ligand
C-24

C-35

SB-612111

NOPC24

-26.08
(0.63)

-24.91
(1.28)

-27.01
(0.65)

NOPC35

-23.17
(1.04)

-27.03
(0.93)

-26.33
(0.67)

NOPSB-612111

-21.65
(0.72)

-22.46
(1.01)

-27.85
(0.56)

Structure

Experimental Docking Results

Binding Score, kJ/mol
Ligand
C-24

C-35

SB-612111 J-113397 J-113397 Trap-101 Trap-101 JTC-801 JTC-801 nor-BNI

Mode I Mode II Mode I Mode II Mode I Mode II

-26.08

-24.91

-27.01

-6.87

-11.06

-13.84

-18.92

-26.12

-29.56

5.53*

Flexible Side
Chains,
-31.11
Water
Sampling

-21.97

-28.41

-23.52

-22.07

-22.44

-21.86

-36.61

-34.12

2.15*

Structure
Rigid

Supplemental Experimental Procedures

Protein Expression and Purification
The human NOP receptor structures presented here use a previously described construct and purification
scheme, with modifications as described below (Thompson et al., 2012). Recombinant baculoviruses were
generated using the Bac-to-Bac system (Invitrogen) and were used to infect Spodoptera frugiperda (Sf9)
insect cells at a density of 2106 cellsml1 at a multiplicity of infection of 5. Infected cells were grown at
27C for 48h before being collected, and the cell pellets were stored at 80C.

Insect cell membranes were disrupted by dounce homogenization of cell pellets in a hypotonic buffer
containing 25mM HEPES, pH7.5, 10mM MgCl2, 20mM KCl and protease inhibitor cocktail (Roche).
Extensive washing of the membranes was performed consecutively by repeated dounce homogenization
and centrifugation in a high osmotic buffer containing 1.0M NaCl, 10mM MgCl2, 20mM KCl and
25mM HEPES, pH7.5 (three times). Purified membranes were resuspended in 500mM NaCl, 20mM
KCl, 50mM HEPES, pH7.5, and 35% (v/v) glycerol, flash frozen with liquid nitrogen, and stored at
80C until further use.

Purified membranes were thawed on ice and incubated with either 25 M C-35 (synthesized by C.
Trapella and R. Guerrini as in (Trapella et al., 2009)) or 50 M SB-612111 (Tocris), in a solution of 500
mM NaCl, 50mM HEPES pH7.5, 20mM KCl, and 5% glycerol (v/v), at 4 C for 60 min. Iodoacetamide
(Sigma) was then added to a final concentration of 1mgml1 and the solution was incubated for another
15min before solubilization with 0.5% (w/v) n-dodecyl--D-maltopyranoside (DDM; Anatrace), and
0.1% (w/v) cholesteryl hemisuccinate (CHS; Sigma) for 3h at 4C. The supernatant was isolated by
centrifugation at 160,000 g for 45min, supplemented with 25mM imidazole, pH7.5, and incubated with

TALON metal ion affinity chromatography resin (Clontech) overnight at 4C. After binding, the resin
was washed with 15 column volumes of buffer 1 (500mM NaCl, 50mM HEPES pH7.5, 20mM KCl,
10mM MgCl2, 5% (v/v) glycerol, 1mM ATP, 25mM imidazole, 25M C-35 or SB-612111, 0.05%
(w/v) DDM and 0.01% (w/v) CHS); and 5 column volumes of wash buffer 2 (buffer 1 without MgCl2 or
ATP), before protein elution with elution buffer (500mM NaCl, 50mM HEPES pH7.5, 20mM KCl,
10% (v/v) glycerol, 250mM imidazole, 25M C-35 or SB-612111, 0.025% (w/v) DDM and 0.005%
(w/v) CHS).

Receptor purity and monodispersity were monitored via SDSPAGE and analytic size exclusion
chromatography. Purified receptor was desalted using a PD midiTrap G-25 column (GE Healthcare) into
a buffer containing 500mM NaCl, 20mM KCl, 5% (v/v) glycerol, 50mM HEPES pH7.5, and 25M C35 or SB-612111. The protein solution was then supplemented with either C-35 or SB-612111 to final
concentrations of 100 M, and further concentrated to 40mgml1 with a 100-kDa molecular mass cut-off
Vivaspin concentrator (GE Healthcare). Typically, protein from 5L of Sf9 biomass was concentrated to
20 L in order to yield a concentrated solution of 40mgml1 appropriate for one round of crystallization.

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