Article

Structures of the entire human opioid receptor

family
Graphical abstract Authors
Yue Wang, Youwen Zhuang,
Jeffrey F. DiBerto, ..., Yi Jiang,
Bryan L. Roth, H. Eric Xu

Correspondence
zhuangyouwen@simm.ac.cn (Y.Z.),
bryan_roth@med.unc.edu (B.L.R.),
eric.xu@simm.ac.cn (H.E.X.)

In brief
Structures of all four human opioid
receptors with the endogenous or
exogenous opioid peptides are reported,
which reveal the conserved mechanisms
of opioid receptor activation and specific
recognition of opioid peptides by the
receptors.

Highlights
d Structures of the entire family of human opioid receptors with
opioid peptides

d The N-terminal ‘‘YGGF’’ message motif of opioid peptides

drives receptor activation

d The C-terminal sequence variations of opioid peptides

address receptor selectivity

d Extracellular loop 2 is key for opioid receptors to filter

specific opioid peptides

Wang et al., 2023, Cell 186, 413–427

January 19, 2023 ª 2022 Elsevier Inc.
https://doi.org/10.1016/j.cell.2022.12.026 ll
 ll

Article
Structures of the entire
human opioid receptor family
Yue Wang,1,2,8 Youwen Zhuang,1,8,* Jeffrey F. DiBerto,3,8 X. Edward Zhou,4 Gavin P. Schmitz,3 Qingning Yuan,1,5
Manish K. Jain,3 Weiyi Liu,1,2 Karsten Melcher,4 Yi Jiang,1,6 Bryan L. Roth,3,* and H. Eric Xu1,2,7,9,*
1The CAS Key Laboratory of Receptor Research and the State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, Shanghai 201203, China

2University of Chinese Academy of Sciences, Beijing 100049, China
3Department of Pharmacology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA
4Department of Structural Biology, Van Andel Research Institute, Grand Rapids, MI 49503, USA
5The Shanghai Advanced Electron Microscope Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai

201203, China
6Lingang Laboratory, Shanghai 200031, China
7School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
8These authors contributed equally
9Lead contact

*Correspondence: zhuangyouwen@simm.ac.cn (Y.Z.), bryan_roth@med.unc.edu (B.L.R.), eric.xu@simm.ac.cn (H.E.X.)

https://doi.org/10.1016/j.cell.2022.12.026

SUMMARY

Opioids are effective analgesics, but their use is beset by serious side effects, including addiction and res-
piratory depression, which contribute to the ongoing opioid crisis. The human opioid system contains four
opioid receptors (mOR, dOR, kOR, and NOPR) and a set of related endogenous opioid peptides (EOPs),
which show distinct selectivity toward their respective opioid receptors (ORs). Despite being key to the
development of safer analgesics, the mechanisms of molecular recognition and selectivity of EOPs to
ORs remain unclear. Here, we systematically characterize the binding of EOPs to ORs and present five
structures of EOP-OR-Gi complexes, including b-endorphin- and endomorphin-bound mOR, deltorphin-
bound dOR, dynorphin-bound kOR, and nociceptin-bound NOPR. These structures, supported by
biochemical results, uncover the specific recognition and selectivity of opioid peptides and the conserved
mechanism of opioid receptor activation. These results provide a structural framework to facilitate rational
design of safer opioid drugs for pain relief.

INTRODUCTION various physiological effects including intrinsic analgesia.14–16

The endogenous opioid system (EOS), composed of four opioid
receptors (ORs) and their endogenous opioid peptide (EOP)
ligands, is an evolutionarily conserved innate response system
for nociception. The EOS is widely distributed in the central and
peripheral nervous systems, as well as in the immune system.1,2
In addition to pain control, the EOS also participates in a broad
range of important biological activities such as reward process-
ing,3,4 perception,5 mood control,6 responses to stress,2,7 regula-
tion of food intake,8 and modulation of immune response.9 ORs
are a group of G protein-coupled receptors (GPCRs), comprising
mOR, dOR, kOR, and nociceptin receptor (NOPR), all of
which transduce signaling primarily through the inhibitory Gi/o pro-
teins.10 ORs can be stimulated not only by EOPs but also by exog-
enously administered opioid compounds, thus blocking pain
signal transduction in both the central and peripheral nervous sys-
tems, resulting in the ultimate analgesic effect.11–13
EOPs, including enkephalins, endorphins, dynorphins, endo-
morphins, and nociceptin, act as neurotransmitters to execute
Nearly all known EOPs are derived from proteolytic cleav-
age of the inactive protein precursors, including proenke-
phalin (PENK), proopiomelanocortin (POMC), prodynorphin
(PDYN), and prepronociceptin (PNOC), by endopeptidases and
carboxypeptidases.1,16 Among them, the well-known b-endor-
phin, which preferentially binds to mOR and dOR, is the proteo-
lytic product of POMC; dynorphins, which have high selectivity
for kOR, are the hydrolysis products of PDYN; and nociceptin/
orphanin FQ (NFQ), which selectively targets NOPR, is the
cleavage product of PNOC.17–19 Endomorphins, such as
endomorphin-1 (YPWF), display high specificity toward mOR
over the other ORs,20 but the precursors of endomorphins
remain unknown.16 A common feature of EOPs is that most of
them share a conserved N-terminal opioid motif of YGGF, except
for NFQ having a specific FGGF motif, to mediate the ‘‘message-
address-efficacy’’ mechanism upon ligand recognition.18,21,22 In
addition to EOPs, ORs also respond to exogenous opioid pep-
tides, such as casomorphin from food or deltorphins from
amphibian skin.23–25

Cell 186, 413–427, January 19, 2023 ª 2022 Elsevier Inc. 413
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Clinically used opioid drugs, which mimic the analgesic func-
tions of EOPs, continue to be the most effective and widely used
therapeutics for analgesia, especially those targeting mOR such
as morphine, oxycodone, and fentanyl. However, their clinical
application is heavily restricted due to the severe side effects
of respiratory depression and addiction, which have led to the
widespread ‘‘opioid crisis’’ in the USA.26,27 Many efforts have
been made to develop effective opioid analgesics without un-
wanted side effects.28 Attention has also been focused on selec-
tive agonists of kOR and dOR, although the development of
opioid drugs targeting kOR and dOR was halted owing to the
serious side effects, including hallucinations associated with
kOR5,29 and convulsions and seizures with dOR.30 It was sug-
gested that the G protein-biased opioids were ideal analgesics
with fewer side effects;28 however, this hypothesis was chal-
lenged by the findings that side effects like respiratory depres-
sion remained in animal studies and clinical trials using G pro-
tein-biased mOR agonists.31,32 Peptide drugs have been
developed to target many GPCRs.33,34 However, the develop-
ment of peptide drugs of ORs with potent pain-relief activities
is hindered by an incomplete molecular understanding of the
interplay among opioid peptides and their receptors.
In addition, selective NOPR agonists and bi-functional ago-
nists acting on both mOR and NOPR have shown promise as
non-addictive analgesics.35,36 However, the mechanism of
NOPR activation remains elusive, impeding the discovery of
NOPR-based opioid drugs. Here, we report human mOR-Gi com-
plex structures bound to the endogenous non-selective peptide
b-endorphin1–31, the longest form of b-endorphin, as well as the
mOR-selective agonist endomorphin-1. To clarify the selective
mechanisms of OR subtype activation, we also determined
structures of the dynorphin A1–13-bound kOR-Gi complex, the
NFQ-bound NOPR-Gi complex, as well as the [D-Ala2] deltorphin
II-bound dOR-Gi complex. (For simplicity, we used b-endorphin,
endomorphin, deltorphin, dynorphin and NFQ referring to
b-endorphin1–31, endomorphin-1, [D-Ala2] deltorphin II, dynor-
phin A1–13, and nociception/orphanin FQ.) These structures pro-
vide molecular insights into how endogenous and exogenous
opioid peptides selectively recognize and activate ORs.
RESULTS
Overall structures of opioid-peptide-bound ORs
We first characterized the binding and activation profiles of the
opioid peptides used in this study for the four human ORs,
mOR, dOR, kOR, and NOPR, through radio-ligand binding assays
and cell-based cAMP inhibition assays combined with biolumi-
nescence resonance energy transfer (BRET) assays to measure
heterotrimeric G protein dissociation. These peptides comprised
b-endorphin, endomorphin, deltorphin, dynorphin, and NFQ.
[D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO), a selective
mOR agonist, was utilized as a reference compound. All of these
peptides are endogenous except for deltorphin; thus, we also
measured the activities of the reference ligands Leu/Met-
enkephalin (Figure S1), which are endogenous but not selective
for dOR. For kOR and NOPR, dynorphin and NFQ used here
served as appropriate endogenous references. In both cAMP
and Gi BRET assays, endomorphin, deltorphin, dynorphin, and
414 Cell 186, 413–427, January 19, 2023
Article
NFQ showed over a hundred times selectivity to their corre-
sponding receptors, mOR, dOR, kOR, and NOPR, respectively,
over other ORs (Figures 1A–1C), which is consistent with radio-
ligand binding data (Figure S1B). However, b-endorphin showed
comparable potency toward mOR and dOR and about ten times
less potency toward kOR and NOPR. These data are consistent
with previous studies on the selective activation of divergent
ORs by these opioid peptides.17,18 We then individually assem-
bled the ORs-Gi signaling complexes with their selective opioid
peptide agonists, purified the complexes to homogeneity, and
determined the structures by using the single-particle cryo-EM
method (Figures 1D–1H and S1). The single-chain antibody
scFv16 was used to stabilize the Gai and Gb interface. The struc-
tures of b-endorphin- and endomorphin-bound mOR-Gi, deltor-
phin-bound dOR-Gi, dynorphin-bound kOR-Gi, and NFQ-bound
NOPR-Gi were determined at the resolution range of 3.0–3.3 Å
(Figures S2 and S3). The EM maps enabled us to unambiguously
model side chains for most portions of ORs, Gi proteins, scFv16
antibody, b-endorphin residues from Y1 to A21, dynorphin resi-
dues Y1 to I8, NFQ residues F1 to L14, and the whole molecules
of endomorphin and deltorphin. Interestingly, we found that both
mOR-Gi and dOR-Gi complexes formed anti-parallel homo-
dimers but with distinct interaction patterns in the dimer inter-
faces (Figure S4A). In the mOR homodimer, the dimer interface
is surrounded by six cholesterols, which interacted with residues
from TM4, TM5, ICL2, and ECL2, with an orientation angle at
about 180 between two monomers (Figure S4B). In contrast,
the dimer interface of the dOR homodimer was mainly packed
through residues from TM5 and TM6 without additional choles-
terols, and the two monomers adopted a clockwise rotation rela-
tive to each other of 160 when measured according to the
conformational change of TM5 (Figure S4C). These anti-parallel
homodimers are presumably not physiologically relevant but
possibly a result of in vitro complex assembly.
Distinct binding of b-endorphin and endomorphin to mOR
As endogenous opioids, endomorphin showed much improved
selectivity and potency to mOR when compared with b-endor-
phin. In our structures, both b-endorphin and endomorphin in-
serted deeply into the orthosteric binding pocket (OBP) with
the N terminus-inside mode, identical to that of DAMGO (Fig-
ure 2A). Despite the fact that the structures of b-endorphin-
and endomorphin-bound mOR are quite similar, with root-
mean-square deviation (RMSD) at 0.4 Å for the Ca atoms of
mOR, the interaction patterns of these two peptides with mOR
are distinct.
b-endorphin formed extensive amphipathic interactions with
mOR from the bottom of the OBP to the top extracellular regions,
including residues from TM1/2/3, TM5/6/7, and ECL1/2. Only
densities of Y1-A21 of b-endorphin could be observed, indi-
cating the flexible conformation of the remaining C-terminal frag-
ment. The N-terminal fragment, known as the ‘‘opioid motif’’
(YGGF), was well overlaid with DAMGO (Figures 2A and 2B).
The opioid motif mediated the majority of interactions with the
bottom OBP within the TMD, whereas the other residues M5-
A21 stretched out of the TMD core and occupied the top region
of the OBP. Residues M5-A21 could further be divided into two
parts: the C-terminal part of P13-A21 formed an a-helix turn at
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Figure 1. Cryo-EM structures of opioid peptide-bound ORs

(A) The pEC50 values of opioid peptides to induce cAMP inhibition in HEK293 cells expressing mOR, dOR, kOR, and NOPR. N.A., not active. The opioid motif is
highlighted in bold.
(B) Dose-dependent response curves of opioid receptors activated by opioid peptide ligands measured by cAMP accumulation assays.
(C) Gi1 dissociation concentration-response curves of peptide ligands at dOR, kOR, mOR, and NOPR. See also Table S2.
(D–H) Cryo-EM maps and corresponding models of the opioid peptide-or-Gi complex, b-endorphin-bound mOR-Gi complex (D), endomorphin-bound mOR-Gi
complex (E), deltorphin-bound dOR-Gi complex (F), dynorphin-bound kOR-Gi complex (G), and NFQ-bound NOPR-Gi complex (H). The densities of respective
ligands have been extracted and shown in surface presentation. See also Figures S2 and S3 and Table S1.

the extracellular end of TM1/2 cleft and the N-terminal part of opioid motif and the C-terminal a-helix turn (Figure 2A). The
M5-T12, which are highly hydrophilic residues and interact extended interactions formed by b-endorphin M5-A21 residues
mainly with ECL2, served as a linker between the N-terminal were absent in DAMGO and the non-peptide agonist

Cell 186, 413–427, January 19, 2023 415

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Figure 2. Binding modes of b-endorphin and endomorphin to mOR

(A) Cross-section of the mOR binding pockets occupied by b-endorphin, endomorphin, and DAMGO.
(B) Structure superposition of b-endorphin with DAMGO (PDB: 8EFQ) and BU72 (PDB: 5C1M) within the mOR OBP revealed similar binding modes of DAMGO and
BU72 relative to the opioid motif.
(C) Polar interactions between b-endorphin and mOR.
(D) Hydrophobic interactions of the opioid motif of b-endorphin with mOR.
(E) Interactions of the C-term a helix of b-endorphin with mOR.
(F) Different binding modes between b-endorphin and endomorphin with W3 in endomorphin overlapping F4 of b-endorphin.
(G) Interactions of F4 in endomorphin with the extended hydrophobic pocket of mOR formed by residues from TM1, TM2, and TM7.
(H) Differential effects of mutations in the extended pocket that accommodates F4 of endomorphin on cAMP inhibition induced by b-endorphin and endomorphin.
Responses were normalized to 100% for the wild type (WT).
(I and J) Mutational effects on cAMP, Gi BRET, and b-arrestin2 recruitment of mOR induced by b-endorphin and endomorphin. Data shown are means ± SEM from
three independent experiments performed in technical duplicates. N.A., not active, refers to both no response and responses that can be observed but for which
reliable parameter estimates cannot be established over the tested concentration range. See also Figure S5 and Tables S3 and S4.

BU72-bound mOR structures.37,38 From the bottom to the top re-
gion of the OBP, the primary amino group in Y1 of the opioid
motif formed a salt bridge (4.5 Å) with D1493.32 (superscript
according to Ballesteros-Weinstein numbering,39 hereafter), of
which the side chain is stabilized by hydrogen-bonds with
Q1262.60 and Y3287.43 (Figure 2B). It is worth noting that such
a polar interaction network is highly conserved in ligand recogni-
tion of ORs.40–44 Indeed, mutations of D1493.32A, Q1262.60A, or
Y3287.43A nearly abolished downstream signaling including Gi
and arrestin signal pathways (Figures 2 and S5; Table S3). The

416 Cell 186, 413–427, January 19, 2023


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main-chain carbonyl of G2 and side chains of E8 and K9
formed hydrogen bonds with W3207.35, R213ECL2/Q214ECL2,
and D218ECL2, respectively (Figure 2C). Mutations of these resi-
dues to the non-polar residue alanine showed compromised
mOR activation by b-endorphin (Figure 2; Table S3), supporting
the binding pose of b-endorphin in our structure. In addition to
polar interactions, at the bottom region, the side chain of Y1
made a hydrophobic interaction network with nearby residues
Y1503.33, M1533.36, V2385.42, I2986.51, and V3026.55. The ben-
zene moiety of F4 was inserted toward TM2/3 and packed
against residues Q1262.60, W135ECL1, V1453.28, and I1463.29 (Fig-
ure 2D). At the top region, the a-helix turn formed hydrophobic
interactions with M671.29, I711.33, L1312.65, and M1322.66
(Figure 2E).
Endomorphin is a tetrapeptide agonist of YPWF that is
different from ‘‘traditional’’ EOPs in its amino acid length and
sequence. In the structure, the tetrapeptide YPWF of endomor-
phin docked into the mOR orthosteric pocket and showed
several differences compared with b-endorphin (Figure 2A).
First, the orientation of the whole peptide was redirected by
the bulky side chain of W3, leading to a closer salt bridge
connection (3.5 Å) between the primary amine group of
Y1 and D1493.32 (Figure 2F). Next, residue W3 but not F4 of
endomorphin engaged in more extensive hydrophobic interac-
tions with the hydrophobic pocket between TM2 and TM3,
similar to that formed by F4 of b-endorphin (Figure 2F). In addi-
tion, F4 of endomorphin formed an extended hydrophobic
pocket composed of residues Y771.39, Y1302.64, H3217.48, and
I3247.39, which was not occupied by the opioid motif of b-endor-
phin, and mutants of the extended pocket have more effects on
endomorphin than b-endorphin (Figures 2G and 2H). Together,
these factors contribute to the higher potency and selectivity of
endomorphin toward mOR than that of b-endorphin.
Unique binding of deltorphin to dOR
Although several structures of dOR bound with either antagonist
or agonist were reported,41,45–46 the molecular features of dOR
activated by subtype-selective opioid peptides were poorly
investigated. The exogenous opioid peptide [D-Ala2] deltorphin
II, referred as deltorphin in this paper, is a derivative of deltor-
phins, the naturally occurring opioid peptides from the skin of
Phyllomedusa species. Deltorphin showed high selectivity
toward dOR over the other ORs.47 The overall structure of deltor-
phin-bound dOR was similar to b-endorphin and endomorphin-
bound mOR (Figure 3A), especially in the intracellular parts of
TM5/6 (Figure 3B), whose conformational changes were pivotal
for activation of GPCRs. Despite the structural similarity, the
OBP of dOR (702 Å3) was smaller than that of mOR (839 Å3)
(Figure 3C).
The whole deltorphin molecule was buried deeply in the OBP
of dOR, with each of its residues forming extensive interactions
with dOR. Recognition of deltorphin in the dOR OBP could be
separated into two regions, of which the N-terminal Y[D-Ala]F
fragment occupied the bottom pocket similar to YGGF of the
opioid motif in mOR and the C-terminal EVVG fragment sat on
the top region formed by the extracellular ends of TM2/6/7 and
ECL2/3 (Figures 3D, 3E, and 3J). In the C-terminal region of del-
torphin, E4 formed close ionic interactions with K2145.39 and
ll
hydrogen-bond interactions with Y1293.33 (Figure 3F). Spatially,
E4 occupied the same space as M5 of b-endorphin, which is
docked into a hydrophobic pocket in mOR (Figure 3F). Thus,
the distinct interaction patterns of E4 in deltorphin and M5 in
b-endorphin might contribute to their selectivity toward dOR
and mOR, respectively. In the C-terminal region of deltorphin,
the side chains of V5 and V6 are orientated toward the highly hy-
drophobic extracellular ends of TM6/7 and ECL3, accommoda-
ting well the hydrophobic pocket formed by residues V2816.55,
W2846.58, L3007.35, H3017.36, and I3047.39 (Figure 3G). In addi-
tion, the main-chain carbonyl of V5 made direct ionic interaction
with R291ECL3, leading to the more inward movement of the
extracellular ends of TM6/7 and ECL3 to the OBP compared
with the reported dOR crystal structures in complex with selec-
tive agonists DPI-287 and KGCHM07 (Figure S4D). Unlike the
bulkier benzamide moiety in DPI-287 or the bistrifluoromethy-
lated benzyl moiety in KGCHM07, the hydrophobic side chains
of V5 and V6 of deltorphin showed less space constraints to
TM6/7, which may facilitate deltorphin to induce inward move-
ment of the extracellular sides of TM6/7 (Figure S4D).
The C-terminal EVVG fragment of deltorphin formed a short
helix below ECL3 of dOR, which contributed to the selectivity
of deltorphin to dOR over mOR (Figure 3H). Structural compari-
son of deltorphin-bound dOR with b-endorphin-bound mOR re-
vealed that the positively charged K3056.58 and the rigid ECL3
of mOR would be energetically unfavorable for deltorphin binding
(Figure 3H). Consistently, mutations of K3056.58I/W or replace-
ment of ECL3 with that of dOR in mOR increased the potency
of deltorphin to induce Gi activation of the mutated mOR receptor
(Figure 3I). Moreover, at the top region of dOR OBP, the main-
chain carbonyl of V6 and the basic amine group of amidated
G7 made additional ionic interactions with K1082.63 and the
main-chain carbonyl of C198ECL2, respectively, facilitating the
potent binding of deltorphin to dOR (Figure 3H).
Full activation state of dOR by deltorphin
Our structure of deltorphin-bound, Gi-coupled dOR revealed
several noticeable differences in ligand-induced conformational
changes at the cytoplasmic regions from the dOR structures
determined by crystallography45,48 (Figure S4D). Compared
with the crystal structure of agonist KGCHM07-bound dOR,77
the intracellular ends of TM5 and TM6 in our structure adopted
a 3.5 Å less outward shift in TM5 and 6.9 Å greater outward
movement in TM6 as measured at the Ca atoms of R2415.66
and K2526.26 (Figure S4G). Moreover, we also observed that
the cytoplasmic end of TM7 of our structure, especially in the
NP7.50xxY motif, adopted an apparent counter-clock rotation
relative to that of KGCHM07-bound dOR. The more pronounced
TM7 rotation might be caused by the distinct rearrangement of
the polar network of the DQY motif (Figure S4F). As a result,
the Y3187.53 in the NP7.50xxY motif swings its side chain upward
near TM6, thus opening the space for the bulky side chain of
R1463.50 from the conserved DRY motif. The much more rotated
NP7.50xxY motif in TM7, together with the reconstruction of the
DRY motif, led to the greater outward movement at the cyto-
plasmic end of TM6 and larger opening of the intracellular cavity
for Gi anchoring (Figure S4H). Thus, we propose that the deltor-
phin-bound dOR structure was in full active conformation,
Cell 186, 413–427, January 19, 2023 417
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Figure 3. Interaction and selectivity of deltorphin toward dOR

(A–C) Structure alignment of b-endorphin-/endomorphin-bound mOR with deltorphin-bound dOR. (A) overall superposition of mOR and dOR structures; (B and C),
structural comparison of the extracellular regions (B) and the cytoplasmic regions (C) of mOR and dOR.
(D) The binding mode of deltorphin in the dOR pocket, which is superposed to that of the YGGY motif of b-endorphin in the mOR pocket. The red dashed circle
indicates the Y[D-Ala]F fragment of deltorphin, which is well overlaid with the YGGF motif in b-endorphin.
(E) Detailed interactions of the N-terminal Y[D-Ala]F fragment of deltorphin with dOR.
(F) Comparison of the interactions of E4 in deltorphin with dOR to the interactions of M5 in b-endorphin with mOR.
(G) Detailed interactions of the C-terminal fragment EVVG of deltorphin with dOR.
(H) Specific interactions of deltorphin with ECL3 and W2846.58 of dOR (pink). Corresponding interactions were not observed in mOR (cyan).
(I) Effects of wild-type and mutated mOR with K3056.58I/W mutations or with dOR ECL3 swap on cAMP inhibition induced by deltorphin. Data shown are means ±
SEM from three independent experiments performed in technical duplicate. N.A., not active, refers to both no response and responses that can be observed but
for which reliable parameter estimates cannot be established over the tested concentration range. See also Figure S5 and Table S5.

whereas the crystal structures, which had less outward move-
ment of TM6 and less rotation of TM7, were in the intermediate
active state.
Recognition of dynorphin by kOR
Dynorphin is an EOP whose N-terminal 13 residue fragment can
activate the OR family except for NOPR (Figures 1A–1C), never-
theless, it has at least 100-fold higher potency toward kOR over
other ORs. In the dynorphin-bound kOR structure, the entire 13
residue peptide is organized vertically into the OBP of kOR, with
only the N-terminal eight residues visible in the EM density map.
This is consistent with the fact that the first eight residue frag-
ments of dynorphin had comparable potency as the N-terminal
13 residue fragment in activation of kOR.21,49 Compared with
the crystal structure of kOR bound to opioid-like agonist
MP1104,50 the dynorphin-bound kOR structure adopts a highly
similar receptor conformation with an RMSD of 0.8 Å for the
entire receptor (Figure 4A). The N-terminal YGGF motif of dynor-
phin occupied the similar bottom pocket of kOR as that by
MP1104. The primary amine group of Y1 mimicked the role of
the tertiary amine group of MP1104, forming an extensive polar
network with the DQY motif and T1112,51 (Figure 4B). Unlike
MP1104, the C-terminal LRRI fragment of dynorphin made addi-
tional interactions with the top region of the kOR OBP, with its

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Figure 4. Recognition of dynorphin by kOR

(A) Structure superposition of dynorphin-kOR with MP1104-kOR (PDB: 6B73).
(B) The conserved polar interactions between DQY motif of kOR with dynorphin and MP1104.
(C) The polar interactions between R6 in dynorphin with kOR.
(D) The interactions between R7-I8 in dynorphin with kOR.
(E) Electrostatic surface representation of the opioid peptides and extracelluar parts of receptors.
(F) The effects of kOR mutations in the extended binding pocket for dynorphin on cAMP inhibition induced by dynorphin. Data shown are means ± SEM from three
independent experiments performed in technical duplicate.
(G) The increased effects of K3056.58E of mOR mutation on cAMP inhibition induced by dynorphin. Data shown are means ± SEM from three independent ex-
periments performed in technical duplicate.
(H) Mutational effects on cAMP, Gi BRET, and b-arrestin2 recruitment of kOR induced by dynorphin. Data shown are means ± SEM from three independent
experiments performed in technical duplicate. N.A., not active, refers to both no response and responses that can be observed but for which reliable parameter
estimates cannot be established over the tested concentration range. See also Figure S5 and Table S6.
(I) Steric clash of Y3137.38 in kOR with V6 in deltorphin and F4 in b-endorphin.
(J) Steric clash of R7 in dynorphin with K3056.58 in mOR and W2846.58 in dOR.
Responses were normalized to 100% for kOR WT.

two positively charged residues R6 and R7 forming direct salt
bridges with E209ECL2 and E2976.58, which are at opposite sides
of the extracellular vestibule of kOR (Figures 4B–4D). Mutations
of E209ECL2 and E2976.58 to alanine or lysine, which disrupted the
ionic interactions, compromised the ability of dynorphin to
induce Gi activation by kOR, corroborating the effect of alanine
substitutions of R6 and R7 of dynorphin52 (Figures 4F and 4H;
Table S6). The more extended interactions of dynorphin with
kOR might explain its high potency for kOR despite the fact
that dynorphin has a relatively flexible N-terminal YGGF motif
compared with the rigid chemical scaffold of the morphinan
agonist MP1104.
The dynorphin-bound kOR structure also explained the
100-fold selectivity of dynorphin for kOR over mOR and dOR.
All three ORs shared the same set of residues in the bottom re-
gions of OBP occupied by their respective subtype-selective

Cell 186, 413–427, January 19, 2023 419

 ll
peptide agonists. However, their extracellular vestibules, which
constituted the top region of the OBP, were non-conserved
and differed in charge distribution, especially in the ECL2/3
and extracellular ends of TM6/7 regions (Figure 4E). A structural
alignment revealed that the extracellular regions of mOR and
dOR, corresponding to the R6–R7 binding pockets in kOR,
were positively charged or highly hydrophobic, which are
incompatible for dynorphin binding. Consistently, the K3056.58E
mutation in mOR increased the potency of dynorphin to mOR by
100-fold (Figure 4G). Moreover, the replacement of ECL2 of
kOR with that of mOR decreased both the potency and activation
efficacy of dynorphin (Figure 4F). The bulky side chain of
Y3137.36 in kOR is predicted to have steric clashes with endo-
morphin and deltorphin, which is highly selective for mOR and
dOR, respectively. It should be noted that the bulky side chains
of ECL3 residues in dOR would have steric clashes with the
C terminus of dynorphin (Figures 4I and 4J). Collectively, we pro-
pose that the ECL2 and extracellular ends of TM6/7 serve as
important selectivity filters of ORs to distinct opioid peptides.
Selectivity of NFQ toward NOPR
NOPR is the last discovered member of the OR family and dis-
plays strict selectivity for its endogenous agonist NFQ over
other opioid peptides. NFQ is a highly basic opioid peptide
(Figure 5A) similar to dynorphin in amino acid composition,
including the C-terminal ‘‘address’’ sequence. Likewise, both
NOPR and kOR contain 30% acidic residues in ECL2 (Fig-
ure 5B), indicating the potentially important role of ECL2 in
the high-affinity binding of NFQ to NOPR rather than mOR
and dOR. However, NFQ differs from all the other known
EOPs in the first N-terminal residue, which is phenylalanine
rather than tyrosine. Y1 is a universal feature of classical
EOPs that participates in a water-mediated polar network
including residues Y3.33 and H6.52 in other ORs, thus facilitating
the stable binding of EOPs.38,48 Nevertheless, the phenylala-
nine substitution of tyrosine in NFQ suggested that Y1 is not
necessary for the high potency of NFQ to NOPR.53
In our structure, NFQ, similar to dynorphin in kOR, is vertically
inserted into the OBP of NOPR, with its N terminus toward the
bottom of the OBP (Figure 5C). The NFQ fragment G6-K14
formed a short stable helix, consistent with a helical conforma-
tion of this region as reported by NMR and modeling studies.54,55
EM densities were clearly observed for the first 14 residues of the
NFQ molecule, consistent with that NFQ1–13 had comparable ac-
tivity with the full-length NFQ.56,51 The FGGF fragment in NOPR
shared a similar conformation with the opioid motif in other ORs
and occupied the corresponding amphipathic bottom region of
the NOPR pocket. The conformation of F1 was stabilized
by hydrophobic interactions with Y1313.33, M1343.36, I2195.42,
V2796.51, and V2836.55, among which Y1313.33 formed p-stack-
ing interactions with the benzene ring of F1 (Figure 5D). Point mu-
tations of these residues largely decreased NFQ-induced NOPR
activation (Figures 5N and S5; Table S7), consistent with the
result of a previous mutagenesis study.42 The primary amine
group of F1, similar to Y1 in other EOPs, formed a salt bridge
with the conserved residue D3.32 from the DQY motif (Figure 5D).
In contrast to the conserved bottom opioid pocket, the extracel-
lular part of NOPR occupied by T5-L14 of NFQ showed great var-
420 Cell 186, 413–427, January 19, 2023
Article
iations from that of the other ORs, contributing to the selectivity
of NFQ toward NOPR. The T5-L14 region of NFQ made both hy-
drophobic and electrostatic interactions with the extracellular
side of NOPR (Figures 5E–5G). The bulky side chains of posi-
tively charged residues of NFQ, including R8, K9, R12, and
K13, stretched toward TM2 and ECL2 regions of NOPR and
formed extensive salt bridge interactions with negatively
charged residues D1102.63, E194ECL2, and E199ECL2(Figure 5F).
Individual mutations of these three residues to alanine dimin-
ished NFQ-induced NOPR activation (Figures 5N and S5;
Table S7).
The ECL2 region is important for the selectivity of kOR and
NOPR to dynorphin and NFQ. Despite the common negatively
charged feature, the distribution of acidic residues of ECL2 in
kOR and NOPR differs, leading to the divergent placement of
the long-extended side chains of basic residues in dynorphin
and NFQ near the ECL2 region. In addition, ECL2 of kOR is three
residues longer and much more inwardly shifted than that of
NOPR. The extra ECL1/2 residues in kOR, including N122ECL1
and R202ECL2, could have steric clashes with the bulky side
chains from the NFQ C terminus, partly accounting for its low af-
finity to NFQ (Figures 5I and 5J). Furthermore, several other non-
conserved residues in the extracellular part of NOPR also play a
vital role in the selectivity of NFQ, including D1102.63 in NOPR,
which makes ionic interaction with R8 of NFQ (Figure 5I). Consis-
tently, the V1182.63D mutation in kOR greatly increased the po-
tency of NFQ to kOR (Figure 5K). Similarly, for mOR and dOR,
the topologies and charge properties of their extracellular sides
formed by ECL2, ECL3, and residue 2.63 may serve as selective
determinants toward NFQ (Figure 5L). For example, dOR has
large, charged residues in ECL3 and greater inward movements
of ECL2 and ECL3, which lead to a narrower extracellular gate
than the corresponding regions of other ORs (Figure 5M), there-
fore preventing the access of longer opioid peptides, such as
NFQ and dynorphin, to the OBP of dOR.
Conserved OR chamber for opioid motif recognition
Canonical EOPs, such as enkephalins, endorphins, and dynor-
phins, follow the formulated ‘‘message-address’’ concept in
amino acid sequences.21,56,57,58 The N-terminal tetrapeptide
YGGF, known as the opioid motif, was hypothesized to be the
‘‘message region’’ and the minimum structural element for
EOP recognition and OR activation. In our structures, both the
YGGF motif from b-endorphin and dynorphin adopt a similar
conformation and occupy a highly aromatic pocket with nearly
the same residue composition from TM2/3/5/6/7 in the bottom
transmembrane part of OBPs in mOR or kOR. Similar to the
YGGF fragment, we observed that YPW of endomorphin, Y
[D-Ala]F of deltorphin, and FGGF of NFQ all share such highly
aromatic pocket in mOR, dOR, and NOPR. Mutations in most
residues around the conserved hydrophobic pocket dramatically
diminish the potency of peptide ligands to their respective
ORs (Figure 6E). In addition, the synthesized YGGF peptide
alone can activate all the four types of ORs with various
potencies (Figure 6F). We thus propose that the common hydro-
phobic pocket in the bottom region of the OBP of ORs serves as
a conserved chamber for opioid ligand recognition and activa-
tion of ORs.
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Article

Figure 5. Selectivity of NFQ toward NOPR

(A) Electrostatic surface representation of NOPR and NFQ.
(B) Sequence alignment of ECL2 of opioid receptors. Positively and negatively charged amino acids are shown in blue and red, respectively.
(C) Structure of NFQ-bound NOPR.
(D–G) Detailed interactions from N terminus to C terminus of NFQ with NOPR. Hydrogen bonds are shown as yellow dashed lines.
(H–J) Superposition of dynorphin-bound kOR with NFQ-bound NOPR showed that ECLs of kOR cause steric clashes with NFQ. (H) Overall steric collision of NFQ
with kOR. (I and J) specific collision of NFQ with kOR in ECL1 (I) and in ECL2 (J).
(K) Effects of kOR mutations with corresponding residues of NOPR on cAMP inhibition induced by NFQ. Data shown are means ± SEM from three independent
experiments performed in technical duplicate.
(L and M) Extracellular view of mOR and dOR shows a steric hindrance with NFQ that prevents NFQ binding, similar to the steric hindrance of NFQ with kOR. The
pink dashed oval highlights the narrower gate that is formed by dOR.
(N) Mutational effects on cAMP, Gi BRET, and b-arrestin2 recruitment of NOPR induced by NFQ. Data shown are means ± SEM from three independent ex-
periments performed in technical duplicate. N.A., not active, refers to both no response and responses that can be observed but for which reliable parameter
estimates cannot be established over the tested concentration range. See also Figure S5 and Table S7.

Consensus activation mechanism of ORs tion mechanism of NOPR. Structural alignment revealed relative
Since the antagonist-bound NOPR structure was available,42 the conformational changes similar to that observed in mOR, dOR,
active NFQ-NOPR structure enabled us to investigate the activa- and kOR, mainly characterized by the inward movement of

Cell 186, 413–427, January 19, 2023 421

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Article

Figure 6. The conserved chambers for opioid peptide recognition in ORs

(A–D) Conformational changes of the conserved motifs during opioid receptor activation. Upper panel: active opioid receptor structures determined in this study.
Lower panel: inactive opioid receptor structures determined previously. mOR (PDB: 4DKL), dOR (PDB: 4N6H), kOR (PDB: 4DJH), and NOPR (PDB: 4EA3).
(E) The fold of changes of EC50 and Emax of mutations on extremely conserved and important residues interacting with the YGGF motif. Data shown are means ±
SEM from three independent experiments performed in technical duplicate. N.A., not active, refers to both no response and responses that can be observed but
for which reliable parameter estimates cannot be established over the tested concentration range.
(F) cAMP inhibition curves of four opioid receptors induced by a synthetic YGGF peptide. Data shown are means ± SEM from three independent experiments
performed in technical duplicate.

TM5 and outward movement of TM6 in their intracellular parts
and clockwise rotations of TM7 in the active NOPR structure
relative to the inactive structure. It was suggested for mOR,
kOR, and dOR that such obvious conformational differences
could be attributed to the ligand-induced collapse of the poten-
tial Na+ pocket and reconstruction of the polar network beneath
the OBP, the toggle switch in CWxP motif, and the rearrange-
ment of the PIF core triad and subsequent conformational
changes in NPxxY motif of TM7 and DRY motif of TM3
(Figures 6A–6D). Superposition of the active structures of all
four subtypes of ORs showed that the residue conformation of
these conserved motifs, which are important for receptor activa-
tion, overlapped well. In addition, the intracellular parts of all ORs
could also be well aligned. These structural observations indi-
cate that the ORs share a similar consensus activation
mechanism.
G protein coupling of ORs
Structure superimposition enabled the elucidation of the
coupling features of ORs with the downstream Gi signaling pro-
tein. The overall structures of OR-Gi complexes are quite
similar to each other, especially the Gi coupling interfaces.
The intracellular parts of Gi coupled ORs were well overlapped
(Figures 7A and 7B), which was expected from the highly
similar activation chamber in OBPs and conserved activation
mechanism of ORs. The interaction interfaces of ORs and Gi
all shared a similar set of residue composition, including resi-
dues in TM3, TM5, TM6, ICL2, and ICL3 of ORs, and residues
in aN, b2-b3 loop, b6, and a5 helix of Gai (Figure S6), as
observed in the DAMGO-bound mOR-Gi structure.38 The a5 he-
lix of Gai inserted into the intracellular cavities of ORs, providing
the major interface module for receptor binding, in a manner
resembling other structures of GPCR-G protein complexes.
The above observations suggested that ORs adopt a highly
conserved mode of coupling to Gi. Two Gi conformational
states, the canonical and non-canonical states, were observed
in previously reported NTSR1-Gi59 and GHSR-Gi structures.60
In our OR-Gi structures, the Gi binding modes are all in the ca-
nonical state as observed in most of the published GPCR-Gi
signaling complexes.

422 Cell 186, 413–427, January 19, 2023


Article
Despite the high similarities in structure and residue composi-
tions in the intracellular regions of ORs, structural differences
were observed in the Gi conformation, characterized by an about
2 Å shift of the a5 helix (Figure 7C) (measured at the Ca atoms of
N331G.H5.03, superscripts refer to the common Ga numbering
[CGN] system, hereafter61) and an about 3.0 Å to 6.0 Å shift of
the aN helix (Figure 7C) (measured at the Ca atoms of
K10G.HN.34). The structural changes of Gi protein varying from
OR subtypes may be due to the subtle conformational differ-
ences in the intracellular ends of TM5/6 and ICL2/3 induced by
opioid peptide ligands.
ORs were known to preferentially activate G proteins of the Gi/o
family over other G protein subtypes, such as Gs and Gq.62 In
addition to the steric effect of the narrow intracellular cavity with
the bulky C terminus of Gas, it was shown that the ICL2 and
ICL3 regions of mOR also served as important filters for its selec-
tivity toward Gi over Gs.38 In our structures, we found that the ICL2
and ICL3 of ORs shared highly similar interaction profiles with Gai.
In ICL2, P34.50 and V/I34.51 formed a hydrophobic network with
L194G.S3.01, F336G.H5.08, T340G.H5.12, I343G.H5.15, and I344G.H5.16
from Gai (Figure 7D). The positively charged ICL3 and the nearby
TM5/6 residues formed ionic patch with the negatively charged
residues from the a4-b6 loop and a5 helix of Gai (Figure 7E). It is
worth noting that the a4-b6 loop is non-conserved in the distribu-
tion of charged residues among Gi, Gs, and Gq (Figures S7A–
S7C); thus, the charged complementation of ICL3 and intracellular
ends of TM5/6 with Gi could partially account for the G protein
selectivity of ORs toward Gi over other G protein subtypes.
DISCUSSION
ORs represent the major drug targets for pain-relief treatment.13
Long-lasting efforts have been made to clarify the structural and
ll
Figure 7. G protein coupling of opioid re-
ceptors
(A) Superposition of four opioid receptors coupled
with opioid peptides and Gi heterotrimer.
(B) Intracellular views of the opioid receptors.
(C) Conformational shift of Ga5 (left) and GaN (right)
of opioid receptor-bound Gai.
(D) Hydrophobic interactions of ICL2 of opioid re-
ceptors with Gai.
(E) Ionic patch of R56.50 and K6.26 of opioid re-
ceptors with Gai.
(F) Alignment of opioid receptor residues that in-
teracted with Gai.
pharmacological understanding of ligand
recognition and receptor activation by
small opioid molecules and related anal-
gesic medications.18,19,63 As endogenous
analgesics of ORs, the EOPs represent
an alternative strategy for drug develop-
ment to manage pain and other diseases
caused by EOS dysfunction, such as
drug abuse and mood disorders. Despite
the fact that the binding of EOPs to ORs
has been widely investigated since the first
discovery of EOPs in the 1970s,16 a structural understanding of
how EOPs preferentially recognize and activate their respective
ORs has remained elusive. In this study, we have reported the
structures of ORs-Gi signaling complexes activated by opioid
peptides, including four EOPs, b-endorphin, endomorphin, dy-
norphin, and NFQ, and the exogenous opioid peptide deltorphin.
Structural comparisons indicated that all the opioid peptides
occupy a highly conserved activation chamber at the bottom
of the OBPs of ORs, which is engaged by the opioid motif,
YGGF. Mutations of residues of the activation chamber dis-
played much more significant effects on receptor activation
compared with other residues in the OBPs, suggesting the indis-
pensable role of the conserved chamber in the recognition and
pharmacological activity of ORs. The extracellular vestibules of
ORs, including ECL2/3 and the extracellular ends of TM2/6/7,
are not conserved in either sequence or charged residue distri-
bution, serving as selectivity filters of ORs for different opioid
peptides and regulating the efficacies of OR activation by opioid
peptides. These structural findings support and extend the
previous message-address hypothesis for EOP binding to
ORs.21,56,57,58 The extracellular vestibule is an important regu-
lator of ligand binding properties of many GPCRs. It was shown
that the electrostatic and conformational differences in the extra-
cellular regions, including ECL2 and ECL3, were critical for the
selective recognition of formyl peptide receptors, FPR1 and
FPR2, toward formyl peptide ligands.64 Moreover, in aminergic
GPCRs, the extended binding pockets located in the less
conserved extracellular vestibules were suggested to be deter-
minants for ligand selectivity and affinity of these receptors.65–67
Therefore, the extracellular vestibule may act as a common regu-
lator for efficient and selective ligand binding of GPCRs.
The structure of NFQ-bound NOPR reported in this study
enabled a comprehensive understanding of the activation
Cell 186, 413–427, January 19, 2023 423
 ll
mechanisms of ORs. Structural comparisons showed that the
important structural motifs related to OR activation, including
the CWxP motif, PIF core triad, sodium pocket, DRY motif, and
NPxxY motif, underwent nearly the same conformational
changes from inactive to active states. The conformations of
these motifs could be superimposed in all four ORs, except
that the NPxxY of dOR has a larger rotation relative to that of
mOR, kOR, and NOPR. These observations indicated that ORs
share a conserved activation mechanism, including the confor-
mational change of toggle switch residue W6.48 and rearrange-
ment of DRY and NPxxY motifs, which ultimately lead to the
outward movement of TM6 for G protein coupling.
ORs show high selectivity toward Gi/o coupling rather than
other G protein subtypes. Structure alignments indicated that
the ICL3 of ORs existed as stable a-helix turn in Gi binding state
(Figure 7E). The stable interaction of ICL3 with Gai is attributed to
not only the extended hydrophobic interactions but also the
electrostatic interactions from the charged patch formed by
the abundant positively charged residues in ICL3 and intracel-
lular ends of TM5/6 to the negatively charged residues of Gai in
a4-b6 loop and a5 helix regions. ICL3 is also visible in many other
GPCR-Gi complexes, including Gi complexes of FPR268 and
CB269,70 (Figure S7A). However, since other G protein isoforms,
such as Gs and Gq/11, do not have the same charge properties as
Gi, even if ICL3 of Gs/Gq/11-coupled receptors have positively
charged amino acids, they cannot form electrostatic interactions
with Gs/Gq/11. This unstable interaction makes the structure
of ICL3 missing in many Gs- and Gq-coupled receptors
(Figures S7B and S7C).71–73 Thus, in addition to ICL2 and the
outward movement of TM6, the specific ionic patch of ICL3 pro-
vides additional selectivity for ORs to couple primarily with Gi/o.
The structures of mOR bound to morphine and fentanyl,74 as
well as with lofentanil and mitragynine pseudoindoxyl,75 have
recently been determined. The overall mOR structures with
different ligands are nearly identical when mOR bound to
morphine, fentanyl, endomorphin, or b-endorphin with the
RMSD of the whole receptor Ca atoms less than 0.4 Å (Fig-
ure S7D). In cAMP assays, fentanyl has greater potency than
DAMGO, whereas endomorphin and b-endorphin are weaker
than DAMGO (Figures S7E and S7F). Structural analysis reveals
that fentanyl binds to an additional pocket of mOR that is not
occupied by endomorphin and b-endorphin, perhaps providing
a higher potency of fentanyl to mOR (Figures S7G and S7H).
Finally, ORs are reported to form homodimers or heterodimers
between receptor subtypes.76,77,79 Although anti-parallel homo-
dimers of mOR and dOR might be not physiologically relevant, it is
interesting to see how these dimers can form in our biochemical
samples. The mOR anti-parallel dimer interface is mediated by
TM4 and TM5 with six cholesterols in between (Figure S4), which
is different from the parallel homodimers observed in mOR crystal
structures.40 The dOR anti-parallel dimer interface is mediated
by TM5 and TM6, similar to the TM5-TM6 dimer interface used
in the parallel dimer of mOR, suggesting that parallel homodimers
of dOR or heterodimers of mOR and dOR could use the same
TM5-TM6 dimer interface.
In summary, we systematically characterized the binding of
several EOPs to all four ORs and determined their Gi complex
structures. Our structures reveal both a conserved pocket for
424 Cell 186, 413–427, January 19, 2023
Article
the opioid YGGF motif and unique structural features of ECL2
and ECL3 for the selectivity of the extended opioid peptides.
The structures also reveal a universal activation mechanism for
all four opioid ORs and specific structural components such as
ICL3 for their selective coupling to the Gi protein subtype.
Together, our work provides a rational framework for under-
standing biology of the opioid system as well as structural tem-
plates for ligand design.
Limitations of the study
There are several limitations in our study. First, the cryo-EM
structures capture relatively static snapshots of ligand binding
poses within the receptors. The dynamic aspects of ligand bind-
ing, for example, the process of ligand entry and release from the
receptor pockets, remain largely unanswered in our study.
Future NMR studies and molecular simulations will be required
to study these dynamic aspects of ORs. Second, the develop-
ment of peptide drugs is limited by instability, low bioavailability,
and poor blood-brain barrier penetration of peptide ligands.
Efforts have been focused on chemical modification of peptide
ligands or specific small molecule ligands. However, due to the
plasticity of ORs, the binding pockets of peptide ligands re-
ported in our structures may not be a good representation for
modified peptide ligands or small molecule ligands. To facilitate
structure-based drug discovery, many more structures with
specific modified peptides or small molecule ligands will be
required.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
B Constructs design
B Preparation of scFv16 antibody
B Expression and purification of opioid receptor- Gi
complexes
B Cryo-EM grid preparation and data collection
B Image processing and 3D reconstruction
B Model building, structure refinement, and figure
preparation
B cAMP accumulation assays
B Radio-ligand binding assays
B Bioluminescence resonance energy transfer assays
B Enzyme-Linked Immunosorbent Assay
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.cell.
2022.12.026.

Article
ACKNOWLEDGMENTS
The cryo-EM data were collected at the Cryo-Electron Microscopy Research
Center, Shanghai Institute of Materia Medica (SIMM), and Advanced Center
for Electron Microscopy, Shanghai Institute of Materia Medica (SIMM). We
sincerely thank all the staff at both institutions for their assistance. This work
was partially supported by grants from the Ministry of Science and Technology
(China) grants (2018YFA0507002 to H.E.X.); the National Natural Science
Foundation of China (82121005 to H.E.X. and Y.J., 32130022 to H.E.X.,
32171187 to Y.J.), the Shanghai Municipal Science and Technology Major
Project (2019SHZDZX02 to H.E.X.); Shanghai Municipal Science and Technol-
ogy Major Project (H.E.X.); the CAS Strategic Priority Research Program
(XDB37030103 to H.E.X.); and the Special Research Assistant Project of Chi-
nese Academy of Sciences (to Y.Z.). B.L.R. is supported by the Michael Hook-
er Distinguished Professorship, the NIMH Psychoactive Drug Screening Pro-
gram and NIH Grant RO1DA055656.
AUTHOR CONTRIBUTIONS
H.E.X. and Y.Z. initialed the project. Y.Z. and Y.W. designed and screened the
expression constructs of hORs and optimized the purification conditions of
protein complexes. Y.W. prepared protein samples of endomorphin-mOR-Gi-
scFv16, deltorphin-dOR-Gi-scFv16, and dynorphin-kOR-Gi-scFv16 com-
plexes for cryo-EM grid making and data collection, performed cryo-EM
structure determination of these protein complexes, performed cAMP assays,
prepared the figures, and participated in manuscript editing. Y.Z. prepared
b-endorphin-mOR-Gi-scFv16 and NFQ-NOPR-Gi-scFv16 complex for cryo-
EM data collection and performed structure determination, prepared the draft
of the manuscript with the input of Y.W., and participated in figure preparation.
J.F.D. performed radio-ligand binding assay, Gi BRET and arrestin BRET as-
says with the assistance of G.P.S. and M.K.J. X.E.Z. modeled and refined all
the structures. Q.Y. assisted in cryo-EM data collection. W.L. assisted with
cellular experiments for the cAMP assay. Y.J. supervised W.L. K.M. super-
vised X.E.Z. and participated in manuscript editing. B.L.R. supervised J.F.D.,
evaluated data and participated in manuscript editing. H.E.X. conceived and
supervised the project and participated in manuscript editing. H.E.X. and
Y.Z. wrote the manuscript with input from Y.W. and B.L.R.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: May 31, 2022
Revised: October 11, 2022
Accepted: December 13, 2022
Published: January 12, 2023
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Article

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
GP64-PE antibody Expression systems Cat# 97-201; RRID: AB_2922960
Anti-HA-FITC antibody, Mouse monoclonal Sigma-Aldrich Cat# H7411; RRID: AB_439704
Monoclonal ANTI-FLAG M2-Peroxidase Sigma-Aldrich Cat# A8592; RRID: AB_439702
(HRP) antibody produced in mouse
Chemicals, Peptides, and Recombinant Proteins
b-endorphin1-31 Genscript Custom Synthesis
Endomorphin-1 Genscript Custom Synthesis
[D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) Med Chem Express Cat# HY-P0210B
[D-Ala2] Deltorphin TFA Med Chem Express Cat# HY-P1013A
Dynorphin A1-13 Genscript Custom Synthesis
Nociceptin/orphanin FQ (NFQ)1-17 Genscript Custom Synthesis
YGGF Genscript Custom Synthesis
Leu-Enkephalin Sigma-Aldrich Cat# E5008
Met-Enkephalin Sigma-Aldrich Cat# M6638
Lauryl maltose neopentyl glycol Anatrace Cat# NG310
Glyco-diosgenin (GDN) Anatrace Cat# GDN101
Cholesteryl Hemisuccinate Anatrace Cat# CH210
ClonExpress II One Step Cloning Kit Vazyme Biotech Co.,Ltd Cat# C112
PrimeSTAR Max Premix TAKARA Cat# R045Q
Protease Inhibitor Cocktail, EDTA-free Bimake Cat# B14003
Apyrase New England Biolabs Cat# M0398L
Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) Sangon Biotech Cat# A600974
Nickel Sepharose resin GE healthcare Cat# 17526801
Anti-Flag resin Smart-Lifesciences Cat# C20042002
FLAG peptide Genscript Custom Synthesis
ESF921 culture medium Expression systems Cat# 96-001-01
Dulbecco’s Modified Eagle Medium (DMEM) Gibco Cat# 12800-017
Fetal Bovine Serum (FBS) Gibco Cat# 10099-141
Hanks’ Balanced Salt Solution (HBSS) Invitrogen Cat# 14065-056
Coelenterazine 400A Nanolight Cat# 340
Coelenterazine-h Promega Cat# S2011
[3H]DADLE PerkinElmer Cat# NET648250U
[3H]DAMGO PerkinElmer Cat# NET902250UC
[3H]Nociceptin PerkinElmer Cat# NET1130050UC
[3H]U-69,593 PerkinElmer Cat# NET952250UC
Critical Commercial Assays
Bac-to-Bac Baculovirus Expression System ThermoFisher Cat# 10359016
LANCE UltracAMP kit PerkinElmer Cat# 2952744
SuperSignal ELISA Pico Chemiluminescent Substrate ThermoFisher Cat# 37069
Deposited Data
mOR-b-endorphin1-31-Gi coordinate This paper PDB: 8F7Q
mOR-endomorphin-1-Gi coordinate This paper PDB: 8F7R
(Continued on next page)

e1 Cell 186, 413–427.e1–e7, January 19, 2023

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Article

Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
2
dOR-[D-Ala ]deltorphin -Gi coordinate This paper PDB: 8F7S
kOR-dynorphin A1-13-Gi coordinate This paper PDB: 8F7W
NOPR-NFQ-Gi coordinate This paper PDB: 8F7X
mOR-b-endorphin1-31-Gi EM map This paper EMDB: EMD-28907
mOR-endomorphin-1-Gi EM map This paper EMDB: EMD-28908
dOR-[D-Ala2]deltorphin-Gi EM map This paper EMDB: EMD-28909
kOR-dynorphin A1-13-Gi EM map This paper EMDB: EMD-28911
NOPR-NFQ-Gi EM map This paper EMDB: EMD-28912
Reagent or Resource
dOR-naltrindole Fenalti et al.45 PDB: 4N6H
dOR-KGCHM07 Claff et al.48 PDB: 6PT2
dOR-DPI-287 Claff et al.48 PDB: 6PT3
50
kOR-MP1104 Che et al. PDB: 6B73
kOR-JDTic Wu et al.43 PDB: 4DJH
mOR-fentanyl-Gi-scFv16 Zhuang et al.74 PDB: 8EF5
mOR-morphine-Gi-scFv16 Zhuang et al.74 PDB: 8EF6
mOR-BU72 Huang et al.37 PDB: 5C1M
mOR-b-FNA Manglik et al.40 PDB: 4DKL
NOPR-Compound-24 Thompson et al.42 PDB: 4EA3
PZM21-mOR-Gi-scFv16 EM map Zhuang et al.74 EMDB: EMD-28086
FPR2-WKYMVm-Gi-scFv16 EM map Zhuang et al.80 EMDB: EMD-20126
Experimental Models: Organisms/strains
E. coli strain OmniMAXTM Invitrogen homemade
TOP10 Competent E. coli TIANGEN Biotech Cat# CB104
Experimental Models: Cell Lines
Spodoptera frugiperda Sf9 cells Expression Systems Cat# 94-001F
HEK-293 cells ATCC Cat# CRL-1573
Recombinant DNA
pFastbac-prolactin-FLAG-mOR(1-388)-His8 This paper N/A
pFastbac-prolactin-FLAG-dOR(2-372, 7 mutations)-His8 This paper N/A
pFastbac-prolactin-FLAG-kOR(3-380)-His8 This paper N/A
pFastbac-prolactin-FLAG-NOPR(2-370)-His8 This paper N/A
pFastbac-DN_Gai(G203A, A326A) This paper N/A
pFastbac-DN_Gai(S47N, G203A, E245A, A326A) This paper N/A
pFastbac-H8-Gb1 This paper N/A
pFastbac-Gg2 This paper N/A
pFastbac-GP67-scFv16-Tev-His8 This paper N/A
pcDNA3.0-HA-FLAG-mOR This paper N/A
pcDNA3.0-HA-FLAG-dOR This paper N/A
pcDNA3.0-HA-FLAG-kOR This paper N/A
pcDNA3.0-HA-FLAG-NOPR This paper N/A
pcDNA3.0-HA-FLAG-mOR-RLuc8 This paper N/A
pcDNA3.0-HA-FLAG-dOR-RLuc8 This paper N/A
pcDNA3.0-HA-FLAG-kOR-RLuc8 This paper N/A
pcDNA3.0-HA-FLAG-NOPR-RLuc8 This paper N/A
pcDNA3.1-Beta3 Addgene Cat# 140988
pcDNA3.1-GGamma9-GFP2 Addgene Cat# 140991
pcDNA3.1-GRK2 Genscript CloneID OHu08965C
(Continued on next page)

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
pcDNA5/FRT/TO-mVenus-Beta-Arrestin2 This paper N/A
pcDNA5/FRT/TO-GAlphai1-RLuc8 Addgene Cat# 140973
Software and Algorithms
Clonemanager Sci-Ed Software http://www.scied.com/pr_cmpro.htm
Prism 8 GraphPad https://www.graphpad.com/scientific-
software/prism/
SerialEM Mastronarde et al.81 http://bio3d.colorado.edu/SerialEM/
MotionCor2 Zheng et al.82 http://msg.ucsf.edu/em/software/motioncor2.html
Relion 3.1 Zivanov et al.83 https://relion.readthedocs.io/en/release-3.1/
Installation.html
UCSF Chimera Pettersen et al.84 https://www.cgl.ucsf.edu/chimera/
UCSF ChimeraX Pettersen et al.85 https://www.cgl.ucsf.edu/chimerax/
Phenix Adams et al.86 https://www.phenix-online.org/
MolProbity Chen et al.87 http://molprobity.biochem.duke.edu/
Coot Emsley et al.88 https://www2.mrc-lmb.cam.ac.uk/personal/
pemsley/coot/
PyMol software Schrödinger https://pymol.org/2/
Adobe Illustrator CC Adobe https://www.adobe.com
Other
100 kDa molecular weight cut-off Amicon Ultra Cat# UFC910024
Quantifoil R1.2/1.3 300-mesh Gold grids Quantifoil https://www.emsdiasum.com/microscopy/
products/grids/quantifoil.aspx
Superdex 200 10/300GL Increase column GE healthcare Cat#28990944
Superose 6 Increase 10/300GL column Cytiva Cat#29091596
PHERAstar FSX with BRET1 and BRET2 optic modules BMG Labtech www.bmglabtech.com

RESOURCE AVAILABILITY

Lead contact
Further information and requests for reagents may be directed and will be fulfilled by the lead contact, H. Eric Xu (eric.xu@simm.
ac.cn).

Materials availability
All stable reagents generated in this study are available from the lead contact without restriction. Plasmids and strains are available
from the authors upon request.

Data and code availability

d Cryo-EM density maps of b-endorphin-mOR-Gi, endomorphin-mOR-Gi, deltorphin-dOR-Gi, dynorphin-kOR-Gi, and NFQ-
NOPR-Gi complex have been deposited in the Electron Microscopy Data Bank (EMDB) under the accession numbers EMD-
28907, EMD-28908, EMD-28909, EMD-28911, and EMD-28912, respectively. Atomic coordinates for the atomic models of
b-endorphin-mOR-Gi, endomorphin-mOR-Gi, deltorphin-dOR-Gi, dynorphin-kOR-Gi, and NFQ-NOPR-Gi complex structures
have been deposited in the Protein Data Bank (PDB) under the accession numbers 8F7Q, 8F7R, 8F7S, 8F7W, and 8F7X,
respectively. These accession numbers are also listed in the key resources table.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Spodoptera frugiperda (Sf9, Expression systems) cells were used for recombinant protein expression. HEK293 cells (ATCC) were
used for functional studies. The Sf9 cells were grown in ESF 921 medium (Expression systems) at 27 C, 120 rpm. HEK293 cells

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were grown in humidified 37 C incubator in condition of 5% CO2. The medium for human cell lines HEK293 was DMEM High-Glucose
(Gibco) containing 10% fetal bovine serum (FBS, Gibco).

METHOD DETAILS

Constructs design
To facilitate expression and receptor anchoring to the membrane, the N-terminus of human opioid receptors were inserted with the
prolactin precursor sequence as a signaling peptide. A FLAG tag was inserted between the signaling peptide and opioid receptors for
purification usage. In addition, the 83His tag was fused at the C-terminus of opioid receptors for two-step purification. For mOR, the
wild type mOR (1-388) truncation was used to obtain the endomorphin-bound mOR-Gi complex, while mOR (1-388) with thermostable
mutation F3.41W was used to prepare the b-endorphin-bound mOR-Gi signaling complex. As for dOR, a total of seven thermostable
mutations, including G731.56V, N902.45S, D952.50G, N1313.35S, S1433.47C, G2686.42V, and A3097.44I, were introduced into the dOR
sequence based on the previously reported dOR structure (PDB: 6PT2).46 The wild-type kOR and NOPR amino acid sequences
were used to prepare the kOR-Gi and NOPR-Gi complexes. These modified opioid receptors were constructed into the pFastBac
vector (ThermoFisher) for expression usage. Two dominant-negative mutations, G203A and A326S, were added to the human
Gai1 (Gai1_2M) to reduce GDP/GTP binding.89 Another two dominant-negative mutations, S47N and E245A, were further incorpo-
rated into Gai1_2M, leading to Gai1_4M.90 All the three components of Gi1 heterotrimer, human Gai1_2M or Gai1_4M, rat His8-Gb1
and bovine Gg2, were constructed into the pFastBac vector (ThermoFisher), respectively.

Preparation of scFv16 antibody

For the expression of scFv16, the GP67 signaling peptide was inserted into the N terminus of scFv16, while a TEV cleavage site-His8
was fused to its C terminus. The modified scFv16 was then subcloned into the pFastBac vector (ThermoFisher). The scFv16 stocks
were prepared by methods previously described.38,91 The purified scFv16 was fast frozen by liquid nitrogen and stored at -80 C for
further usage in GPCR-Gi signaling complex preparation.

Expression and purification of opioid receptor- Gi complexes

Opioid receptors, Gai1, Gb1, and Gg2 were co-expressed in sf9 insect cells using the Bac-to-Bac baculovirus expression system
(ThermoFisher). The sf9 cells were cultured in ESF 921 serum-free medium (Expression Systems). For expression of opioid re-
ceptor-Gi signaling complexes, baculoviruses of each opioid receptor, Gai1, Gb1, and Gg2 were added in equal proportions to
the cell culture when the cell density reached 43106 cells/mL. For mOR, dOR and NOPR, we used Gai1_2M for the expression of
protein complexes, while for kOR, we used the Gai1_4M instead of Gai1_2M to form the stable signaling complex. After being
cultured for 48 hours, the cells were harvested by centrifugation at 1300 g for 20 min and frozen at -80  C for further protein
purification.
The cell pellets were thawed at room temperature and resuspended in 20 mM HEPES pH 7.2, 50 mM NaCl, 10mM KCl, 5 mM
MgCl2, 0.3 mM TCEP, protease inhibitor cocktail (Bimake, 1mL/100mL suspension). After quick cell lysis by the French press, the
cell suspension was centrifugated at 100,000 3 g for 30 min at 4 C. The cell precipitates were then collected and resuspended in
buffer including 20 mM HEPES pH 7.2, 75 mM NaCl, 5 mM CaCl2, 5 mM MgCl2, 5% glycerol, 0.3 mM TCEP, protease inhibitor
cocktail (Bimake, 1mL/100mL suspension). The b-endorphin or endomorphin-bound mOR-Gi complexes were assembled on cell
membranes by the addition of 20mM b-endorphin or 10mM endomorphin. For the dOR-Gi, kOR-Gi, and NOPR-Gi complexes,
10mM deltorphin, 10mM dynorphin, and 10mM NFQ were added to stimulate the formation of signaling complexes, respectively.
The protein complexes were all assembled at room temperature. Half an hour after ligand addition, the cell suspension was subse-
quently treated with 25mU mL 1 apyrase to digest the nucleotides. The suspension was continually incubated for another one hour at
room temperature before being extracted from the membrane using 0.5% (w/v) lauryl maltose neopentylglycol (LMNG, Anatrace) and
0.1% (w/v) cholesteryl hemisuccinate TRIS salt (CHS, Anatrace). After membrane solubilization for 3 hours at 4 C, the solubilized
fractions were isolated by centrifugation at 100,000 3 g for 45 min and then incubated overnight at 4 C with pre-equilibrated
Nickel-NTA resin using distilled deionized water (dd H2O). After batch binding, the nickel resin with immobilized protein complex
was manually loaded onto a gravity flow column. The nickel resin was firstly washed with 20 column volumes of 20 mM HEPES,
pH 7.2, 100 mM NaCl, 25 mM imidazole, 0.3 mM TCEP, 0.05% LMNG (w/v), 0.01% CHS (w/v) and 10 mM corresponding ligands,
and then eluted with the same buffer containing 300 mM imidazole.
The Ni-NTA eluates were further incubated plus 2.5 mg scFv16 by batch binding with 2 mL FLAG resin (Smart-Lifesciences) for
2 hours at 4 C. The FLAG resin embedded with protein complexes was then washed by 10 column volumes of detergent buffer
containing 20 mM HEPES, pH 7.2, 100 mM NaCl, 0.3 mM TCEP, 0.0075% LMNG (w/v), 0.0025% GDN, 0.002% CHS (w/v),
10 mM ligands. Subsequently, the material bound to FLAG resin was then eluted with the same detergent buffer above containing
200mg/mL FLAG peptide. The complex was then concentrated using an Ultra Centrifugal Filter (Millipore, Sigma, 100 kDa molecular
weight cutoff) and subjected to Superdex 200 Increase 10/300 GL (GE Healthcare) or Superose 6 Increase 10/300 GL (GE Healthcare)
size exclusion chromatography columns pre-equilibrated with buffer containing 20 mM HEPES, pH 7.2, 100 mM NaCl, 0.00075%
LMNG (w/v), 0.00025% GDN, 0.0002% CHS (w/v), 0.3 mM TCEP and 5 mM ligands. Fractions of the main peak of the protein com-
plexes were collected and concentrated to approximately 10-13 mg/ml for electron microscopy experiments.

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Cryo-EM grid preparation and data collection

Before grid making, the monodispersed b-endorphin-mOR-Gi complex, endomorphin-mOR-Gi complex, deltorphin-dOR-Gi complex,
dynorphin-kOR-Gi complex, and NFQ-NOPR-Gi complex were concentrated to about 7.5 mg mL-1, 6.8 mg mL-1, 13 mg mL-1, 12 mg
mL-1, and 10 mg mL-1, respectively. For cryo-EM grids preparation, about 3.0 mL of each protein complex sample was applied
individually to EM grids (Quantifoil R1.2/1.3 holey carbon films, 300 mesh Au) that were glow-discharged for 50s in a Vitrobot cham-
ber (FEI Vitrobot Mark IV). Vitrification was performed in 100% humidity at 4 C. The sample was blotted for 3s before plunge-freezing
into liquid ethane. The prepared cryo-EM grids were stored in liquid nitrogen for screening and data collection usage.
For the b-endorphin-mOR-Gi complex and dynorphin-kOR-Gi complex, automatic cryo-EM movie stacks were collected on an FEI
Titan Krios microscope operated at 300kV accelerating voltage in Cryo-Electron Microscopy Research Center, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences (Shanghai, China). The microscope was operated with a nominal magnification of
81,0003 in super-resolution counting mode. A total of 4,959 movies for the dataset of b-endorphin-mOR-Gi-scFv16 complex and
4,254 movies for the dataset of dynorphin-kOR-Gi-scFv16 complex were collected individually by a Gatan K3 Summit direct electron
detector equipped with a Gatan energy filter (operated with a slit width of 20 eV) (GIF) using the SerialEM software.81 The images were
recorded at a dose rate of 23.3 e/Å2/s with a defocus ranging from -0.5 to -3.0 mm. The total exposure time was 3 s and intermediate
frames were recorded in 0.083 s intervals, resulting in a total of 36 frames per micrograph.
For the deltorphin-dOR-Gi-scFv16 complex and NFQ-NOPR-Gi-scFv16 complex, automatic cryo-EM movie stacks were collected
on an FEI Titan Krios microscope operated at 300kV in Advanced Center for Electron Microscopy, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences (Shanghai, China). The microscope was equipped with a Gatan Quantum energy filter.
The movie stacks were collected automatically using a Gatan K3 direct electron detector with a nominal magnification of
105,0003 in super-resolution counting mode at a pixel size of 0.42 Å. The energy filter was operated with a slit width of 20 eV.
Each movie stack was dose-fractionated in 36 frames with a dose of 1.39 electrons per frame and collected within a defocus ranging
from -0.8 to -1.8 mm. The total exposure time was 2.35 s. A total of 6,841 movies were collected for deltorphin-dOR-Gi-scFv16 com-
plex. A total of 7,125 movies were collected for NFQ-NOPR-Gi-scFv16 complex. Data collection was performed using EPU with one
exposure per hole on the grid squares.
For the endomorphin-mOR-Gi-scFv16 complex, automatic cryo-EM movie stacks were collected by a Titan Krios G4 at 300KV
accelerating voltage equipped with Falcon4 detector in Advanced Center for Electron Microscopy, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences (Shanghai, China). The movie stacks were collected automatically with a nominal
magnification of 96,0003 in counting mode at a pixel size of 0.8 Å. Each movie stack was dose-fractionated in 160 frames with
50 total doses (e/ Å2) and collected within a defocus ranging from -1.0 to -2.0 mm. A total of 6,418 movies for the dataset of endo-
morphin-mOR-Gi-scFv16 complex were collected. Data collection was performed using EPU with one exposure per hole on the
grid squares.

Image processing and 3D reconstruction

Movie stacks were subjected to beam-induced motion correction using MotionCor 2.1.82 Contrast transfer function (CTF) parameters
for each non-dose-weighted micrograph were determined by Ctffind4.92 Automated particle selection and data processing were per-
formed using RELION-3.1 beta283 or RELION-4.0 beta.93
For the datasets of b-endorphin-mOR-Gi-scFv16 and dynorphin-kOR-Gi-scFv16 complex, the movie stack was aligned, dose
weighted, and binned by 2 to 1.071 Å per pixel. The micrographs with resolution worse than 4.0 Å and micrographs imaged within
the carbon area of grid squares were abandoned, producing 3,908 and 3,523 micrographs respectively to do further data processing.
For the b-endorphin-mOR-Gi-scFv16 complex, template-based particle selection yielded 3,095,570 particles which were subjected
to reference-free 2D classifications to discard bad particles. The cryo-EM density map of the FPR2-Gi complex78 (EMDB: EMD-
20126) low-pass filtered to 40 Å was used as a reference model for four rounds of maximum-likelihood-based 3D classifications.
Further 3D classification focusing on the anti-parallel dimer of receptors produced 320,047 particles. These particles were subse-
quently subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a density map with an indicated global
resolution of 3.2 Å at a Fourier shell correlation of 0.143. For dynorphin-kOR-Gi-scFv16 complex, template-based particle selection
yielded 2,480,199 particles which were subjected to reference-free 2D classifications to discard bad particles. The map of the FPR2-
Gi complex (EMDB: EMD-20126) low-pass filtered to 40 Å was used as a reference model for four rounds of maximum-likelihood-
based 3D classifications, resulting in three well-defined subsets with 173,555 particles. These particles were subsequently subjected
to 3D refinement, CTF refinement, Bayesian polishing, and post-processing, which generated a density map with an indicated global
resolution of 3.3 Å at a Fourier shell correlation of 0.143.
For the datasets of endomorphin-mOR-Gi-scFv16 complex, the movie stack was aligned, dose weighted, and binned by 2 to 1.6 Å
per pixel. The micrographs with resolution worse than 4.0 Å and micrographs imaged within the carbon area of grid squares were
abandoned, producing 5,704 micrographs to do further data processing. Template-based particle selection yielded 2,012,939
particles which were subjected to reference-free 2D classifications to discard bad particles. The map of PZM21-mOR-Gi-scFv1674
low-pass filtered to 60 Å was used as a reference model for four rounds of maximum-likelihood-based 3D classifications. Further
3D classification focusing on the anti-parallel dimer of receptors produced 185,378 particles. These particles were subsequently sub-
jected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a density map with an indicated global resolution
of 3.3 Å at a Fourier shell correlation of 0.143.

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For the datasets of deltorphin-dOR-Gi- scFv16 complex and NFQ-NOPR-Gi-scFv16 complex, the movie stack was aligned, dose
weighted and binned by 2 to 0.84 Å per pixel. The micrographs with resolution worse than 4.0 Å and micrographs imaged within the
carbon area of grid squares were abandoned, producing 6,413 and 6,439 micrographs respectively to do further data processing
micrographs to do further data processing. For the deltorphin-dOR-Gi- scFv16 complex, template-based particle selection yielded
4,839,714 particles which were subjected to reference-free 2D classifications to discard bad particles. The map of PZM21-mOR-Gi-
scFv16 low-pass filtered to 40 Å was used as a reference model for three rounds of maximum-likelihood-based 3D classifications.
Rotary anti-parallel dimer conformations were observed. The further 3D classification was performed with a mask excluding one of
the G protein heterotrimers with poor density. The final 3D classifications lead to two well-defined subsets with 650,970 particles.
These particles were subsequently subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a density
map with an indicated global resolution of 3.0 Å at a Fourier shell correlation of 0.143. For NFQ-NOPR-Gi-scFv16 complex, template-
based particle selection yielded 4,809,226 particles which were subjected to reference-free 2D classifications to discard bad parti-
cles. The map of dynorphin-kOR-Gi-scFv16 complex (this study) low-pass filtered to 60 Å was used as a reference model for four
rounds of maximum-likelihood-based 3D classifications resulting in one well-defined subset with 184,353 particles. These particles
were subsequently subjected to 3D refinement, CTF refinement, and Bayesian polishing, which generated a density map with an indi-
cated global resolution of 3.3 Å at a Fourier shell correlation of 0.143.

Model building, structure refinement, and figure preparation

The previously published structures of mOR-Gi complex (PDB: 8EF5), dOR (PDB: 6PT2), kOR (PDB: 6B73), and NOPR (PDB: 4EA3)
were used as the start models for model building and refinement of mOR-Gi, dOR-Gi, kOR-Gi, and NOPR-Gi, respectively. The struc-
tural model was firstly docked as a rigid body into the cryo-EM density maps using UCSF Chimera.84 Models were then manually
rebuilt and/or adjusted in COOT.88 Real space and Rosetta refinements were performed using Phenix.86 The model statistics
were validated using MolProbity.87 Structural figures were prepared in ChimeraX85 and PyMOL (https://pymol.org/2/). The final
refinement statistics are provided in Table S1. The maximum distance cutoffs for polar hydrogen-bond interactions and hydrophobic
interactions were set at 3.5 Å and 4.5 Å, respectively.

cAMP accumulation assays

The open reading frames of the full-length human opioid receptors (hORs) were cloned into the pcDNA3.0 vector. For detecting Gi
signaling of mOR, dOR, kOR, and NOPR, HEK293 cells expressing WT or mutant hORs were harvested and resuspended in HBSS
buffer (Gibco) containing 0.1% BSA, 5mM HEPES (Gibco) and 500 mM IBMX (Abcone) at a density of 6 3105 cells/ mL. Cells
were then plated onto 384-well assay plates at 3000 cells/ 5 mL/ well. Another 5 mL buffer containing 2 mM Forskolin and various con-
centrations of test ligands were added to the cells. After incubation at room temperature for 30 minutes, the intracellular cAMP level
was tested by a LANCE Ultra cAMP kit (PerkinElmer, TRF0264) and EnVision multiplate reader according to the manufacturer’s
instructions.

Radio-ligand binding assays

Binding assays were performed on crude membrane preparations made from HEK293T cells transiently transfected with dOR, kOR,
mOR, or NOPR. The following radioligands (final concentration in nM) were used to label dOR, kOR, mOR, and NOPR, respectively:
[3H]DADLE (0.5 nM), [3H]U-69,593 (0.5 nM), [3H]DAMGO (0.5 nM), [3H]nociceptin (0.5 nM). In brief, 75 mL of membrane were co-incu-
bated with 25 mL of 5x radioligand and 25 mL of 5x drug for 90 minutes in standard assay buffer (50 mM TRIS hydrochloride buffer,
pH 7.4, containing 10 mM magnesium chloride and 0.1 mM EDTA) supplemented with 1 mg/mL fatty-acid free bovine serum albumin
and 0.1 mg/mL ascorbic acid. Following incubation, membranes were collected onto GF/C glass fiber filter-bottom, polyethylenei-
mine-coated 96-well microplates by vacuum filtration (an initial vacuum followed by three subsequent vacuums separated by washes
with cold harvesting buffer (50 mM Tris-HCl, pH 7.4). Non-specific binding for dOR, kOR, mOR, and NOPR were determined by 10 mM
concentrations of deltorphin-II, dynorphin A, DAMGO, and NFQ, respectively. All data were analyzed using GraphPad Prism.

Bioluminescence resonance energy transfer assays

Bioluminescence resonance energy transfer (BRET) assays were performed to measure G protein dissociation and beta-arrestin
recruitment. For G protein dissociation measurements, the TRUPATH platform94 was used as experiments were performed as pre-
viously described.94,95 In brief, wildtype or mutant dOR kOR, mOR, or NOPR were co-transfected with Gai1-RLuc8, Gb3, and GFP2-
Gg9 in a 1:1:1:1 ratio in HEK293T cells maintained in Dulbecco’s Modified Eagle Media (DMEM) supplement with 10% fetal bovine
serum (FBS) and 1% penicillin-streptomycin (pen-strep). After a minimum of 12 hours, cells were plated in 96-well microplates in
DMEM supplemented with 1% dialyzed FBS and 1% pen-strep. After an additional minimum of 12 hours, plates were first vacuum
aspirated and 60 mL of assay buffer (1x Hank’s Balanced Salt Solution in phosphate buffered saline, 20 mM HEPES, pH 7.4) were
added to the wells. Next, 10 mL of coelenterazine 400a diluted in assay buffer (50 mM) were added to the wells, and allowed to incu-
bate for 5 minutes. Next, 30 mL of 3x drug diluted in drug buffer (assay buffer supplemented with 3 mg/mL fatty acid-free bovine serum
albumin and 0.3 mg/mL ascorbic acid) were added to the wells, and allowed to incubate for 5 minutes. Last, plates were read using a
BMG Labtech PHERAstar FSX with BRET2 plus optic module. For b-arrestin recruitment measurements, RLuc8 was cloned directly
into the end of the C-terminus of wildtype and mutant dOR kOR, mOR, or NOPR, and experiments were performed as previously

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described96,97 and identically to BRET2 assays with the following exceptions: 1) Wildtype or mutant kOR-RLuc8, mOR-RLuc8, or
NOPR-RLuc8 were co-transfected with GRK2 and mVenus-b-arrestin2 in a 1:1:5 ratio and dOR-RLuc8 was co-transfected with
GRK2 and mVenus-b-arrestin2 in a 1:1:15 ratio, 2) 10 mL of coelenterazine h diluted in assay buffer (50 mM) were added to the wells
instead of coelenterazine 400a, and 3) plates were read using a BMG Labtech PHERAstar FSX with BRET1 plus optic module. All data
were analyzed using GraphPad Prism.

Enzyme-Linked Immunosorbent Assay

Cell surface expression of the opioid receptor-RLuc8 constructs used in the BRET-based b-arrestin2 recruitment assays were
measured using an enzyme-linked immunosorbent assay (ELISA). Cells were transfected and plated in 96-well microplates as
described for the BRET assays. For the ELISA, all steps were performed at room temperature, with care given to liquid addition
and aspiration to minimize cell loss. First, plates were vacuum aspirated and 50 mL of 4% paraformaldehyde (PFA) in phosphate buff-
ered saline (PBS) were added to the wells and allowed to incubate for 10 minutes. Next, the PFA was aspirated, and cells were
washed twice with 50 mL of PBS. After the second PBS wash, 50 mL of 5% bovine serum album in PBS (blocking buffer) were added
to the wells and allowed to incubate for 30 minutes. Following incubation, an additional 25 mL of blocking buffer containing mono-
clonal ANTI-FLAG M2-Peroxidase (HRP) antibody produced in mouse (1:10,000 final dilution) were added to the wells and allowed
to incubate for 1 hour. Following incubation, cells were washed twice with 50 mL of PBS. Finally, 50 ml of SuperSignal enzyme-linked
immunosorbent assay (ELISA) Pico Chemiluminescent Substrate were added to the wells, and luminescence was counted using a
BMG Labtech PHERAstar FSX.

QUANTIFICATION AND STATISTICAL ANALYSIS

All functional study data were analyzed using Prism 8 (GraphPad) and presented as means ± S.E.M. from at least three independent
experiments. Non-linear curve fit was performed using a three-parameter logistic equation [log (agonist vs response)] for cAMP and
BRET assays. Competitive binding was performed using a one site – Fite Ki equation.

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Supplemental figures

Figure S1. Purification of ORs-Gi complexes bound to respective opioid peptides, related to Figure 1
(A) Sequence alignment of common opioid peptides. Conserved residues are indicated in red.
(B) Characterization of the activities of opioid peptide ligands on all four ORs by radio-ligand competition binding assays.
(C) G protein BRET assays of all four opioid receptors induced by peptide ligands.
(D–H) Size exclusion chromatography (SEC) profiles (left) and SDS-PAGE analysis (right) of purified opioid peptide-bound ORs-Gi complexes.
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Figure S2. Structure determination of b-endorphin- and endomorphin- bound mOR-Gi complexes and dynorphin- bound kOR-Gi complex,
related to Figures 1, 2, and 4
(A) Representative cryo-EM raw image and 2D classification averages of the b-endorphin-mOR-Gi complex.
(legend continued on next page)
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(B) The flow chart of cryo-EM data processing of the b-endorphin-mOR-Gi complex.
(C) The Fourier shell correlation (FSC) curve of the b-endorphin-mOR-Gi complex. The global resolution of the final 3D density map defined at the FSC = 0.143
is 3.22 Å.
(D) Local resolution and angle distribution maps of the b-endorphin-mOR-Gi complex.
(E) EM density of transmembrane helices of mOR, a5 helix of Gai1, and b-endorphin.
(F) Representative cryo-EM raw image and 2D classification averages of the endomorphin-mOR-Gi complex.
(G) The flow chart of cryo-EM data processing of the endomorphin-mOR-Gi complex.
(H) The FSC curve of the endomorphin-mOR-Gi complex. The overall resolution of the final 3D density map defined at the FSC = 0.143 is 3.28 Å.
(I) Local resolution and angle distribution map of the endomorphin-mOR-Gi complex.
(J) EM density of transmembrane helices of mOR, a5 helix of Gai1, and endomorphin.
(K) Representative cryo-EM image and 2D classification averages of the dynorphin-kOR-Gi complex.
(L) The flow chart of cryo-EM data processing of the dynorphin-kOR-Gi complex.
(M) The FSC curve of the dynorphin-kOR-Gi complex. The global resolution of the final 3D density map defined at the FSC = 0.143 is 3.34 Å.
(N) Local resolutions and angle distribution map of the dynorphin-kOR-Gi complex.
(O) EM density of transmembrane helices of kOR, a5 helix of Gai1, and dynorphin.
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Figure S3. Structure determination of deltorphin-bound dOR-Gi and NFQ-bound NOPR-Gi complexes, related to Figures 1, 3, and 5
(A) Representative cryo-EM micrographs of deltorphin-bound dOR-Gi complex.
(B) The flow chart of cryo-EM data processing of the deltorphin-bound dOR-Gi complex. In the procedure of data processing, two types of complexes with good
densities on the receptor part were divided. One of the two classes contained well-resolved G protein density, but with fewer particles. The overall resolution of
the complete dOR-Gi map was determined at 3.03 Å. For the other type with more particles, the density on G protein part was poorly resolved and the overall
resolution was determined at 2.62 Å.
(C and D) 2D classification averages and the FSC curve of the complete deltorphin-bound dOR-Gi complex. At the FSC 0.143 cutoff, the overall resolution of the
final 3D density map is defined at 3.03 Å.
(legend continued on next page)
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(E and F) 2D classification averages and the FSC curve of the incomplete deltorphin-bound dOR-Gi complex. At the FSC 0.143 cutoff, the overall resolution of the
final 3D density map is 2.62 Å.
(G) Local resolution and angle distribution maps of the complete deltorphin-bound dOR-Gi complex.
(H) EM density of transmembrane helices of dOR, a5 helix of Gai1, and deltorphin.
(I) Representative cryo-EM image and 2D classification averages of the NFQ-NOPR-Gi complex.
(J) The flow chart of cryo-EM data processing of the NFQ-bound NOPR-Gi complex.
(K) Local resolutions and angle distribution map of the NFQ-bound NOPR-Gi complex.
(L) The FSC curve of the NFQ-bound NOPR-Gi complex. At the FSC 0.143 cutoff, the overall resolution of the final 3D density map is defined at 3.28 Å.
(M) Local EM density of transmembrane helices of NOPR, a5 helix of Gai1, and NFQ.
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Figure S4. Full activation of the dOR-Gi complex by deltorphin, related to Figure 3
(A) Different rotatory angles and interaction patterns of mOR and dOR anti-parallel homodimers. Upper panel: mOR-Gi complex; lower panel: dOR-Gi complex.
(B) The mOR anti-parallel dimer interacted mainly through the interface of TM4 and TM5, the cholesterol molecules at the dimer interface are shown in black stick
representation.
(C) dOR dimer rotated at about 159 . The dimer interface was mainly formed through the residues from TM5 and TM6.
(D) Superposition of deltorphin-bound dOR with previous reported crystal structures of active and inactive dOR (PDB: 4N6H; 6PT2; 6PT3).
(E) Different binding modes of three agonists in the dOR OBP.
(F) Superposition of deltorphin-dOR with KGCHM07-dOR showed closer interactions of deltorphin with the DQY motif in dOR. Red dashes indicate hydrogen
bonds of deltorphin with the DQY motif. Black dashes indicate hydrogen bonds of KGCHM07 with DQY motif. Color usage: deltorphin-bound dOR, pink;
KGCHM07 bound dOR, green; deltorphin: teal; KGCHM07: yellow.
(G) Structural comparison of deltorphin and KGCHM07 bound dOR in TM5, TM6, and TM7-H8 regions. Larger inward movement of TM5, outward movement of
TM6, and rotation of TM7 were observed in deltorphin-bound dOR structure relative to that bound to KGCHM07.
(H) Pronounced rearrangement of the DRY and NPxxY motifs in the deltorphin-bound dOR complex relative to the KGCHM07-dOR complex.
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Figure S5. cAMP, Gi BRET, and b-arrestin2 recruitment assays of wild-type opioid receptors and mutants, related to Figures 2–6 and
Tables S4–S7
HEK293 cells were transfected with WT or mutant opioid receptors. Cell-based cAMP, Gi BRET, and b-arrestin2 recruitment assays were performed. Results of
each point were represented as mean ± SEM from at least three independent experiments and each in duplicate. Dose response curves were further analyzed
using the ‘‘log(agonist) vs. response—three parameter’’ function in Graphpad Prism.
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Figure S6. Interactions between opioid receptors and Gai, related to Figure 7
(A–D) Representative interactions of four opioid receptors with Gai. The left panel displays interactions with a5. The middle panel shows ICL2 interactions with aN
and b2-b3 loop. The right panel shows ICL3 interactions with b6 and a5. The electrostatic surface of ICL3 is also displayed.
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Figure S7. GPCRs coupled to different G protein subtypes, related to Figure 7

(A) Interactions of ICL3 of ORs with Gai showing the positive residues of ICL3 to form electrostatic interactions with the negative charge patch of Gai. Similar ionic
patches were also observed in Gi subtype-selective GPCRs such as FPR2 and CB2.

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(B and C) ICL3 of most Gaq/11- or Gas-coupled receptors were missing in their structures.
(D) Structural comparisons of mOR bound to morphine (PDB: 8EF6), fentanyl (PDB: 8EF5), b-endorphin and endomorphin.
(E and F) cAMP response of mOR induced by morphine, fentanyl, b-endorphin and endomorphin, relative to DAMGO.
(G and H) Superposition of the binding modes of fentanyl with b-endorphin and endomorphin, showing the extra-interactions of fentanyl with mOR (circle).