Structure of the nociceptin/orphanin FQ receptor in
complex with a peptide mimetic
Aaron A. Thompson1*, Wei Liu1*, Eugene Chun1*, Vsevolod Katritch1, Huixian Wu1, Eyal Vardy2, Xi-Ping Huang2,
Claudio Trapella3, Remo Guerrini3, Girolamo Calo4, Bryan L. Roth2, Vadim Cherezov1& Raymond C. Stevens1
Members of the opioid receptor family of G-protein-coupled
nervous system, where they have key roles in nociception and anal-
gesia. Unlike the ‘classical’ opioid receptors, d, k and m (d-OR,
k-OR and m-OR), which were delineated by pharmacological
criteria in the 1970s and 1980s, the nociceptin/orphanin FQ
(N/OFQ) peptide receptor (NOP, also known as ORL-1) was dis-
covered relatively recently by molecular cloning and characteriza-
tion of an orphan GPCR1. Although it shares high sequence
similarity with classical opioid GPCR subtypes ( 60%), NOP has
a markedly distinct pharmacology, featuring activation by the
endogenous peptide N/OFQ, and unique selectivity for exogenous
ligands2,3. Here we report the crystal structure of human NOP,
solved in complex with the peptide mimetic antagonist compound-
24 (C-24) (ref. 4), revealing atomic details of ligand–receptor recog-
nition and selectivity. Compound-24 mimics the first four amino-
ing of these peptides. The X-ray structure also shows substantial
conformational differences in the pocket regions between NOP and
the classical opioid receptors k (ref. 5) and m (ref. 6), and these are
probably due to a small number of residues that vary between these
receptors. The NOP–compound-24 structure explains the divergent
selectivity profile of NOP and provides a new structural template for
the design of NOP ligands.
The pharmacological effects of NOP are complex and distinct from
classical opioid receptors. N/OFQ shares sequence similarity with
A, but does not interact with d-OR, k-OR or m-OR. Similarly, the
classical opioid peptides have very low affinity for NOP. Unlike the
classical opioid receptors, NOP is also insensitive to most morphine-
like small molecules including naloxone, thereby yielding a pharma-
cologically important discriminatory feature between NOP and
the NOP system has important roles in the control of central and
peripheral functions including pain, anxiety and mood, food intake,
learning and memory, locomotion, cough and micturition reflexes,
Understanding the structural requirements for NOP ligand selectivity
and modes of binding is therefore paramount for the optimization of
future agonist- and antagonist-based therapeutics.
The 3.0A˚resolution X-ray crystal structure of the human NOP
receptor in complex with C-24 was determined by replacing 43
N-terminal residues of the receptor with thermostabilized apocyto-
chrome b562RIL (BRIL)8, and by truncating 31 carboxy-terminal
residues of NOP (Fig. 1a) (see Methods). We found that this BRIL–
NOP fusion is functional and responds to N/OFQ and the small
molecular agonist SCH-221510 (ref. 9), activating endogenous
heterotrimeric Gi/Goproteins in HEK293T cells, albeit with reduced
on the basis of the pronounced thermostability it imparts on the
receptor (Supplementary Fig. 1), its high affinity (half-maximum
inhibitory concentration (IC50)50.27nM) and antagonist potency
(IC5050.1nM) for NOP, and its selectivity ($1,000-fold)4.
system, where it antagonizes N/OFQ effects on nociception10and
produces beneficial responses in experimental models of Parkinson’s
disease11. The NOP structure revealed C-24 binding deep within the
domain of N/OFQ (Phe1-Gly2-Gly3-Phe4), a similar sequence to
that of canonical opioid peptides (Tyr1-Gly2-Gly3-Phe4)7,12(Sup-
plementary Fig. 2).
variation of the extracellular module with boundaries defined by
proline-induced kinks13. NOP contains five such kinks in the seven-
transmembrane core located at residue positions Pro1052.58,
Pro1844.59, Pro2275.50, Pro2786.50and Pro3167.50(superscripts indi-
yielding repercussions on the shape of the ligand-binding pocket.
Notably,the extracellulartip ofhelix V in NOP is shiftedbymorethan
4A˚as compared with the k-OR5and m-OR6crystal structures (Protein
Data Bank (PDB) accessions 4DJH and 4DKL, respectively), thereby
residues 184 and 215) and an expansion of the orthosteric pocket
(Supplementary Fig. 5). However, compared with the chemokine
receptor CXCR4, the extracellular tip of helices VI and VII are tilted
extracellular half of helix I in NOP is pulled in towards the axis of the
This alternative conformation of helix I is facilitated by the presence of
flexible glycine residues located at an apparent ‘hinge point’ that are
Specifically, the backbone of ECL1 in NOP is nearly indistinguishable
from that of k-OR and CXCR4. ECL2 forms an elongated b-hairpin,
which is tethered to the extracellular tip of helix III by a structurally
conserved disulphide bond between Cys1233.25and Cys200ECL2. This
b-hairpin motif is also observed in k-OR and CXCR4, suggesting a
common structural motif of the c-branch16class A peptide-binding
*These authors contributed equally to this work.
and Division of ChemicalBiologyand Medicinal Chemistry,Universityof North CarolinaChapel Hill Medical School,Chapel Hill, North Carolina27599, USA.3DepartmentofPharmaceutical Sciencesand
of Neuroscience, University of Ferrara, 44121 Ferrara, Italy.
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receptors. Unlike d-OR and m-OR, the ECL2s of k-OR and NOP are
entrance to their binding pocket very acidic (Supplementary Fig. 6).
it and differences in charge distribution possible determinants for
selectivity. These details are consistent with N/OFQ–dynorphin A
chimaera peptide data showing that replacement of six residues on
the address domain of N/OFQ with the corresponding residues from
dynorphin A markedly impaired affinity and activity towards NOP17.
Intracellular loop (ICL) 2 of NOP receptor molecule ‘B’ in the
asymmetric unit forms a short a-helix, which has been observed in
many other GPCRs (Fig. 1c); the ICL2 is tethered to the seven-
transmembrane core via a salt bridge between Arg162ICL2found in
all opioid receptors and Asp1473.49from the conserved ‘D(E)RY’
motif (Supplementary Fig. 7). The ICL3 connecting helices V and VI
rearrangements in these helices and binding of heterotrimeric Gi/Go
proteins. Therefore, the structure of ICL3 found in the NOP receptor
molecule ‘A’, which has 15 residues in a coil conformation and a
hydrogen bond between the backbone carbonyl of Val245ICL3
and the Arg 2596.31side chain, is probably just one of the possible
configurations the loop can adopt. Structural alignment with
thermostabilized A2AAR (PDB accession 3PWH)18also suggests
V and VI are shorter and further apart than in A2AAR, and the coiled
part of ICL3 is longer than A2AAR (15 residues in NOP versus 8
residues in A2AAR) (Fig. 1c and Supplementary Fig. 7).
The NOP–C-24 structure highlights specific residues in the pocket
that are essential for N/OFQ binding and receptor subtype selectivity
(Fig. 2). The orthosteric binding pocket of NOP is relatively large,
reflecting its ability to bind large endogenous peptides. With a similar
pose in both NOP molecules (root mean squared deviation
several hydrophobic and electrostatic interactions. Mutagenesis of the
binding pocket of NOP defined the relative impact of specific residues
on C-24 and N/OFQ binding and function (Supplementary Tables 4
and 5). The protonated nitrogen of the C-24 piperidine ring forms a
crucial salt bridge with Asp1303.32— a residue that is conserved in the
opioid receptor family and all biogenic amine GPCRs. Mutations of
Asp1303.32to either alanine or asparagine abrogate N/OFQ binding,
highlighting the requirement of the negative charge at this position19
(Fig. 2 and Supplementary Tables 4 and 5), and it has been proposed
by residues fromhelices III, V and VI. The benzofuran ‘head’ group is
sandwiched between Met1343.36and Tyr1313.33, in which the Met3.36
side chain adopts a different, more buried rotamer as compared to
k-OR, thereby allowing a deeper penetration of the C-24 ring system.
This is consistent with the modest effect of a Met1343.36Ala mutation
on the potency of NOP ligands (Supplementary Tables 4 and 5). A
Tyr1313.33Phe mutation had no effect on agonist binding, whereas
Tyr1313.33Ala was deleterious (Supplementary Tables 4 and 5),
suggesting that Tyr131 participates in p-stacking interactions with
Phe1 of the peptide19.
in C-24 binding and a more than 300-fold reduction in N/OFQ
potency, and mutation of Tyr3097.43abolishes binding of C-24 and
reduces N/OFQ potency ,7-fold (Supplementary Tables 4 and 5).
Interestingly, both Gln2.60and Tyr7.43are present in the k-OR struc-
ture, albeit in very different conformations (Supplementary Fig. 8).
The crystal structure of NOP in complex with C-24 afforded us a
unique opportunity to determine the molecular basis for both the
pronounced subtype selectivities (Fig. 3). Notably, we verified that the
of C-24 to the NOP receptor, with an r.m.s.d. of ,0.9A˚. Moreover,
docking of another piperidine derivative, compound-35 (C-35)22,
closely mimics the binding of C-24, whereas docking of a less active
stereoisomer compound-36 (ref. 22) yields a considerably distorted
binding pose in the pyrrolidine region and a reduced binding score
(not shown). C-24 has previously been proposed22to mimic the
N-terminal four residues of N/OFQ-related peptide antagonists
and UFP-101 (ref. 24). Automated docking of the four N-terminal
residues of UFP-101 results in a conformation of the Nphe1-Gly
2-Gly3-Phe4 tetrapeptide in which the Nphe1 and Phe4 rings of the
peptide make the same hydrophobic interactions as the aromatic rings
of C-24, and the N-terminal amino group forms a salt bridge with
Asp1303.32, thus supporting the proposed similarity in the binding
poses between small molecules and peptide analogues (Fig. 3c).
The ‘address’ domain of N/OFQ (residues 5–17) was previously
shown by NMR to have a strong preference for a-helical secondary
structure25,26, which is probably preserved in UFP-101 as the only
difference in this domain are the mutations Leu14Arg and Ala15Lys.
Docking of the full-length UFP-101 suggests a plausible fit of the
a-helical address domain into the binding pocket entrance shaped
Figure 1 | Structural overview of the NOP receptor. a, NOP (grey) is shown
in ribbon representation with its ECL2 highlighted (red). The bound ligand
C-24 is depicted as green spheres, and transparent disks highlight the
extracellular (EC) and intracellular (IC) membrane boundaries (coloured blue
k-OR (PDB accession4DJH)5andCXCR4 (PDBaccession3ODU)15, coloured
grey, yellow, blue and orange, respectively. Compared with k-OR, the
extracellular portion of helix I from NOP is tilted inwards towards the
orthosteric pocket, in a similar conformation to CXCR4. c, Structural
superposition of NOP molecules A and B and thermostabilized A2AAR (PDB
accession3PWH)18, colouredgrey, yellow andgreen, respectively,highlighting
conformational differences between the ICLs.
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residues of the peptide forming ionic interactions with acidic side
chains of NOP (Fig. 3c–e).
Interactions of the address domain of N/OFQ(1–13) with helices II
(residues 107–113)27and VII (residues 296–302)19were previously
demonstrated by photocrosslinking, a finding consistent with our
mutagenesis data showing the crucial importance of Asp1102.63in
the binding of N/OFQ but not small molecule antagonists or the
agonist SCH-221510 (Supplementary Table 5). These results suggest
a similar binding mode for the address domains of N/OFQ-derived
peptides. On the other hand, note that the k-OR-binding peptide
dynorphin A has a Pro10 in the middle of the address sequence12that
is unfavourablefora-helixformation, suggesting potential differences
in the binding mode for this classical opioid peptide.
As mentioned earlier, NOPdisplays markedly reduced affinities for
morphine-based small molecules and the classical opioid receptor
peptide ligands: N/OFQ contains an N-terminal FGGF instead of
theYGGFmotif foundinthe classicalopioid receptorpeptideligands.
Previous biochemical studies attributed this distinct selectivity profile
to the three residuepositions in the binding pocket of NOP that differ
fromall other opioid receptors: Ala2165.39(Lys in others), Gln2806.52
(His in others) and Thr3057.39(Ile in others). Mutation of these three
positions on the N/OFQ receptor to classical opioid receptor residues
hasbeen shown to be sufficient for conferring high-affinity binding to
a dynorphin-derived k-OR selective peptide28,29. Moreover, the same
three mutations conferred nanomolar-range NOP binding of
morphine-based opioid antagonists such as bremazocine, naltrexone
(nor-BNI)29. The crystal structures of NOP and k-OR show that the
side chains of these three residues are pointing towards the interior of
the binding pocket (Fig. 4 and Supplementary Fig. 8). In NOP,
Gln2806.52and Thr3057.39are involved in C-24 interactions, and all
three of the cognate residues at these positions are involved in k-OR
nor-BNI antagonists5. Notably, although most of the modified side
chains are polar, none form direct hydrogen bonding interactions to
the ligands tested, so that the selectivity profiles cannot be explained
by simple polar-to-hydrophobic (or vice versa) changes of ligand
that several of the NOP-specific side-chain changes, including two of
the substitutions mentioned earlier (Ala5.39Lys and Gln6.52His), are
involved in a large-scale reshaping of the binding pocket and an
alternative coordination of water molecules (Fig. 4). Located closer
and is accompanied by an outwards shift of the extracellular half of
helix V in the NOP crystal structure, and an inwards shift of helix VI.
Opioid receptor subtype alteration of the large Lys5.39side chain and
the accompanying shifts of the a-helices reshape the entrance to the
pocket, and this probably affects the binding of address domains of
peptides and synthetic ligands.
The k-OR structure reveals a cluster of water molecules that is
coordinated by two of the classical opioid receptor-specific residues
Figure 2 | The orthosteric ligand-binding pocket. a, Cartoon representation
of NOP with its large orthosteric ligand-binding pocket shown as a blue
transparent surface. ECL2 is coloured red in all subsequent figures.
b, Extracellular view of the pocket with bound C-24 depicted as green sticks.
hydrogen bond interactions and salt bridges. d, A sA-weighted 2mFo2DFc
electron density map contoured at 1.0s (0.0173eA˚23) around C-24 inside the
ligand-binding pocket. e, Schematic representation of C-24 interactions with
NOP (B), with labelled distances (A˚). Residue labels are coloured according to
the effect on C-24 binding when replaced with alanine. Magenta-labelled
no effect on the binding of C-24, although it is crucial for N/OFQ binding.
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water molecule is coordinated by His6.52and seems to preclude a
buried rotamer conformation of Met3.36in k-OR that is observed in
the side chain in these two crystal structures (Fig. 4c). This Met3.36
residue is conserved in all opioid receptors and makes extensive
hydrophobic interactions with the corresponding ligands in both
NOP and k-OR. As a consequence, the 7-hydroxyisoquinoline head
group of the k-OR ligand JDTic is not able to penetrate as deeply into
this area of the orthosteric pocket as compared with the benzofuran
group of C-24. The ‘reoriented’ hydroxylated head group of JDTic is
stabilized by a hydrogen bond interaction to a water molecule that
is coordinated by the backbone carbonyl of Lys2275.39, potentially
explaining the need for a tyrosine residue at the N terminus of
dynorphin A. With modifications of Lys5.39to Ala5.39and His6.52to
Gln6.52in the NOP receptor, remodelling of the binding pocket that
and water rearrangements provides a likely explanation for selectivity
in the message domain of the peptide ligands.
Perhaps most intriguing are the evolutionary differences between
NOP and the other three classical opioid receptors (k-OR, m-OR and
d-OR). Despite high sequence identity between receptors, marked
differences in ligand selectivity between these opioid receptors go in
This situation is very different from other GPCR subfamilies (for
example, b-adrenergic and muscarinic) in which different subtypes
signal by the same ligands via highly conserved orthosteric pocket
architectures. With structural data for k-OR5, m-OR6and NOP now
available, and the fourth (d-OR) opioid receptor structure likely to
come in the near future, one can begin to investigate the ligand struc-
ture activity relationships and evolutionary aspects of this receptor
subfamily in greater detail.
Figure 4 | Conformational differences in the ligand-binding pocket
between NOP–C-24 and k-OR–JDTic. a, ‘Sliced’ surface representation of
NOP, highlighting the deep binding pocket bound with C-24 (coloured green)
and JDTic (coloured magenta) from the superimposed k-OR structure.
b, c, Different views of NOP (coloured grey with green C-24) superimposed
with the k-OR structure5(PDB accession 4DJH; coloured blue with magenta
lines for NOP and k-OR, respectively. The water molecules from the k-OR
structure are depicted as cyan spheres. Residue labels are coloured black and
blue for NOP and k-OR, respectively. The conformational shifts observed
between helices V and VI that result in different binding pocket architectures
are highlighted in b. The alternative rotamer of Met3.36in the pocket (134 in
NOP and 142 in k-OR), which affects the orientation of the head group of the
ligand, is highlighted in c.
Nphe 1Nphe 1
Phe 4 Phe 4
Figure 3 | Molecular docking in the orthosteric-binding pocket. a–e, The
docking of C-24 (a), its analogue C-35 (b) and peptide antagonist UFP-101
(c–e) in the NOP. The crystallographic pose of C-24 is green in all panels, and
the docked molecules (C-24, C-35 and UFP-101) are coloured yellow. The
Nphe1-Gly2-Gly3-Phe4 tetrapeptide portion of the docked UFP-101 is
depicted as sticks, and the ‘address’ domain (residues 5–17) of this peptide is
represented as a cartoon. A ‘sliced’ side-view of the pocket is shown in c, and a
view from the extracellular surface is shown in d. e, The electrostatic surface
potentials of the UFP-101 peptide, coloured blue to red, corresponding to
positive and negative surface potentials (13 to 23kTe21), respectively. ECL2
is coloured red, and the acidic Asp and Glu residues from the ECL2 b-hairpin
are depicted as red sticks.
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BRIL–NOP was expressed in Spodoptera frugiperda (Sf9) insect cells. Ligand-
binding asays were performed as described in Methods. Sf9 membranes were
solubilized using 0.5% (w/v) n-dodecyl-b-D-maltopyranoside and 0.1% (w/v)
cholesteryl hemisuccinate, and purified by immobilized metal ion affinity
(LCP) method. The protein–LCP mixture contained 40% (w/w) concentrated
receptor solution, 54% (w/w) monoolein and 6% (w/w) cholesterol. Crystals were
grown in 40nl protein-laden LCP bolus overlaid by 0.8ml of precipitant solution
(25–30% (v/v) PEG400, 100–200mM potassium sodium tartrate tetrahydrate,
100mM Bis-Tris propane, pH6.4) at 20uC. Crystals were collected directly from
the LCP matrix and flash frozen in liquid nitrogen. X-ray diffraction data were
collected at 100K on the 23ID-B/D beamline (GM/CA-CAT) of the Advanced
Photon Source at the Argonne National Laboratory using a 10-mm collimated
minibeam. Diffraction data from 23 crystals were merged for the final data set.
Data collection, processing, structure solution and refinement are described in
Methods. Flexible docking of small molecules and peptides was performed with
the ICM molecular modelling package (Molsoft LLC).
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 22 February 2011; accepted 30 March 2012.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements This work was supported by PSI:Biology grant U54 GM094618
for biological studies and structure production, NIH Roadmap grant P50 GM073197
for technology development and R01 DA017204, R01 DA27170, and the NIMH
of University (FIRB Futuro in Ricerca 2010 grant to C.T.). We thank J. Francis for
suggesting the idea to pursue the NOP receptor; J. Velasquez for help on molecular
biology;T.Trinh, K.Allin andM. Chu for helponbaculovirus expression; A. Walkerand
E. Abola for assistance with manuscript preparation; J. Smith, R. Fischetti and
N.Sanishviliforassistance indevelopmentand useoftheminibeam and beamtimeat
GM/CA-CAT beamline 23-ID at the Advanced Photon Source, which is supported by
National Cancer Institute grant Y1-CO-1020 and National Institute of General Medical
Sciences grant Y1-GM-1104.
Author Contributions A.A.T. optimized the constructs, purified and crystallized the
receptor in LCP, optimized crystallization conditions, grew crystals for data collection,
collected the data and refined the structure, and prepared the manuscript. W.L.
assays, collected diffraction data, and assisted with preparing the manuscript. E.C.
and prepared the manuscript. H.W. assisted with membrane preparations, provided
advice on crystallization strategies, and assisted with preparing the manuscript. E.V.
and X.-P.H.performedligand-bindingandsite-directedmutagenesisstudies. C.T., R.G.
pharmacological studies, and assisted with preparing the manuscript. B.L.R.
V.C. assisted with the crystallization in LCP, collected diffraction data, processed
diffraction data, and prepared the manuscript. R.C.S. was responsible for the overall
project strategy and management and wrote the manuscript.
Author Information The coordinates and the structure factors have been deposited in
the Protein Data Bank under accession code 4EA3. Reprints and permissions
information is available at www.nature.com/reprints. The authors declare competing
financial interests: details accompany the full-text HTML version of the paper at
www.nature.com/nature. Readers are welcome to comment on the online version of
this article at www.nature.com/nature. Correspondence and requests for materials
should be addressed to R.C.S. (firstname.lastname@example.org).
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Cloning, expression and purification. NOP contains a ,50-amino-acid
extracellular domain at its N terminus, with a relatively high content of leucine
and proline residues (26% and 14%, respectively) and three putative N-linked
glycosylation sites. Despite high thermostability in the presence of select small
N terminus were unsuccessful. Although deletion of the C terminus (NOP-DC; 31-
amino-acid deletion) resulted in increased expression, any truncation of the N
terminus decreased the expression levels. However, replacement of the N terminus
with several soluble fusion proteins restored the expression to levels that were com-
to as BRIL–NOP in the manuscript) that was crystallized to 3.0A˚resolution in
complex with the non-peptide antagonist C-24 (Banyu Pharmaceuticals).
The wild-type human NOP gene (encoded by OPRL1; UniProt accession
P41146) was synthesized by DNA2.0 with codon optimization for Spodoptera
frugiperda (Sf9), and then cloned into a modified pFastBac1 vector (Invitrogen)
by a Flag tag at the N terminus, and a PreScission protease site followed by a
103His tag at the C terminus. Thirty-one amino acids were deleted from the C
NOP were replaced with the thermostabilized apocytochrome b562RIL from
extension PCR30. Recombinant baculoviruseswere generatedusing the Bac-to-Bac
system (Invitrogen) and were used to infect Sf9 insect cells at a density of 23106
cellsml21at a multiplicity of infection of 5. Infected cells were grown at 27uC for
48h before being collected, and the cell pellets were stored at 280uC.
in a hypotonic buffer containing 25mM HEPES, pH7.5, 10mM MgCl2, 20mM
was performed consecutively by repeated dounce homogenization and centrifu-
gationinthesame hypotonicbuffer(approximately oncemore),followedbyhigh
osmotic buffer containing 1.0M NaCl, 10mM MgCl2, 20mM KCl and 25mM
HEPES, pH7.5 (three to four 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 280uC until further use.
(synthesized by C. Trapella and R. Guerrini), 500mM NaCl, 20mM KCl, 50mM
(Sigma) was then added to the membranes at a final concentration of 1mgml21
for another 15min before solubilization with 0.5% (w/v) n-dodecyl-b-D-
maltopyranoside (DDM; Anatrace), and 0.1% (w/v) cholesteryl hemisuccinate
(CHS; Anatrace or Sigma) for 3h at 4uC. The supernatant was isolated by
centrifugation at 160,000g for 45min, supplemented with 25mM imidazole,
pH7.5, and incubated with TALON metal ion affinity chromatography resin
(Clontech) overnight at 4uC. Typically, 0.75ml of resin (slurry) per 1l of original
culture volume was used. After binding, the resin was washed with 15 column
volumes of wash buffer 1 (500mM NaCl, 20mM KCl, 10mM MgCl2, 50mM
HEPES, pH7.5, 5% (v/v) glycerol, 1mM ATP, 25mM imidazole, 25mM C-24,
(same as wash buffer 1, but without ATP and MgCl2), before protein elution with
elution buffer (500mM NaCl, 20mM KCl, 50mM HEPES, pH7.5, 10% (v/v)
glycerol, 250mM imidazole, 25mM C-24, 0.025% (w/v) DDM and 0.005% (w/v)
CHS). Purified receptor was exchanged into a buffer containing 500mM NaCl,
midiTrap G-25 column (GE Healthcare). BRIL-DN-NOP-DC was then supple-
mented with C-24 to a final concentration of 100 mM, and concentrated from
,0.4mgml21to 40mgml21with a 100-kDa molecular mass cut-off Vivaspin
concentrator (GE Healthcare). Receptor purity and monodispersity was followed
using SDS–PAGE and analytical size exclusion chromatography.
Pharmacological assays. The differentNOP constructs (codon optimizedfor Sf9
expression) were cloned from pFastBac into pCDNA3.1 and expressed in
HEK293T cells. Mutations (Q107A, D110A, D130A, Y131A, M134A, I219A,
Q280A and Y309A) were introduced into the NOP sequence using standard
QuikChange protocols. Binding affinity was determined from competition bind-
ing assays using3H-N/OFQ as a radioligand. NOP receptor-mediated inhibition
ofthecyclic AMP response wasmeasured usinga cAMP biosensor(see ref. 31for
mutations within 3.5A˚from the antagonist C-24 in the structure. HEK293T cells
were transiently transfected for binding assays or functional assays. Antagonist
inhibition response curves were measured in the presence of a concentration of
agonist (N/OFQ or SCH-221510) approximately corresponding to its EC80value
(the concentration that leads to an 80% maximum response). Results were
analysed using GraphPad Prism.
Crystallization. Protein samples of BRIL-DN-NOP-DC (concentrated to
40mgml21) in complex with C-24 were reconstituted into thelipidiccubic phase
(LCP) by mixing with molten lipid using a mechanical syringe mixer32. The
protein–LCP mixture contained 40% (w/w) protein solution, 54% (w/w)
monoolein (Sigma) and6%(w/w)
Crystallization trials were performed in 96-well glass sandwich plates33
(Marienfeld) by the NT8-LCP (Formulatrix) or mosquito LCP (TTP LabTech)
crystallization robots using 40nl protein-laden LCP bolus overlaid with 0.8ml
precipitant solution in each well, and sealed with a glass coverslip. Protein recon-
stitution in LCP and crystallization trials were carried out at room temperature
(,20–22uC). The crystallization plates were stored and imaged in an incubator/
average size of 4031033mm were obtained within ,14 days in 25–30% (v/v)
PEG400, 100–200mM potassium sodium tartrate tetrahydrate, 100mM Bis-Tris
23ID-B/D beamline (GM/CA CAT) of the Advanced Photon Source at the
Argonne National Laboratory, using a 10mm collimated minibeam. Because of
1u oscillation and 3–10s exposure before moving to a fresh part of the crystal, if
scaled and merged together using HKL2000 (ref. 34).
was obtained by Phaser35using the receptor domain of the DN-k-OR-T4L-DC/
JDTicstructure(PDB accession 4DJH)5andthe thermostabilized apocytochrome
b562RIL protein (PDB accession 1M6T) as search models. With two antiparallel
receptor molecules in the asymmetric unit of the P21lattice, one of the BRIL
domains is disordered whereas the second forms crystal lattice contacts with
is flexible owing to the presence of a flexible linker and the absence of any crystal
Phenix37. The initial stages of refinement were performed with simulated anneal-
ingandrebuildinginto composite omitmaps, andnoncrystallographic symmetry
and translation/libration/screw (TLS) refinement were implemented throughout.
Figures were created using PyMOL38, and electrostatic surface potentials were
obtained using APBS39.
Molecular modelling of C-24 analogues and UFP-101 peptide binding to
NOP. Docking of high-affinity NOP specific ligands was performed using an
modelling package as described previously40. Internal coordinate (torsion) move-
ments were allowed in the side chains of the binding pocket, defined as residues
within 10A˚distance of C-24 in the crystal structure. Other side chains and the
backboneofthe protein were kept as inthecrystal structure. An initial conforma-
tion for small molecule ligands was generated by Cartesian optimization of the
ligandmodel in MerckMolecularForce Field. Docking wasperformed by placing
and global conformational energy optimization of the complex40,41. To facilitate
Monte Carlo procedure used ‘soft’ van der Waals potentials and high Monte Carlo
gradually decreasing temperature. A harmonic ‘distance restraint’ was applied
between the protonated amine (of piperidine group in the small ligand or Nphe1
in the UFP-101 peptide) and the carboxyl of the Asp1303.32side chain in the initial
With UFP-101, the first six residues Nphe1-Gly2-Gly3-Phe4-Thr5-Gly6 were
conformation for the rest of the peptide (Ala7-Arg8-Lys9-Ser10-Ala11-Arg12-
Lys13-Arg14-Lys15-Asn16-Gln17). At least 10 independent runs of the docking
procedure were performed for each NOP-ligand. The docking results were consid-
ered ‘consistent’ when at least 80% of the individual runs resultedin conformations
performed on a 12-core Linux workstation.
cholesterol (AvantiPolar Lipids).
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