An investigation into the origin of the biased agonism associated with the urotensin II receptor activation

Article abstract of DOI:10.1002/psc.2740

The urotensin II receptor (UTR) has long been studied mainly for its involvement in the cardiovascular homeostasis both in health and disease state. Two endogenous ligands activate UTR, i.e. urotensin II (U-II) and urotensin II-related peptide (URP). Exte

Full text of DOI:10.1002/psc.2740

Special Issue Article
Received: 30 September 2014
Revised: 9 December 2014
Accepted: 11 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2740
An investigation into the origin of the
biased agonism associated with the
urotensin II receptor activation**
Diego Brancaccio,a‡ Francesco Merlino,a‡ Antonio Limatola,a
Ali Munaim Yousif,a Isabel Gomez-Monterrey,a Pietro Campiglia,b
Ettore Novellino,a Paolo Griecoa,c and Alfonso Carotenutoa*
The urotensin II receptor (UTR) has long been studied mainly for its involvement in the cardiovascular homeostasis both in health
and disease state. Two endogenous ligands activate UTR, i.e. urotensin II (U-II) and urotensin II-related peptide (URP). Extensive
expression of the two ligands uncovers the diversified pathophysiological effects mediated by the urotensinergic system such as
cardiovascular disorders, smooth muscle cell proliferation, renal disease, diabetes, and tumour growth. As newly reported, U-II
and URP have distinct effects on transcriptional activity, cell proliferation, and myocardial contractile activities supporting the idea
that U-II and URP interact with UTR in a distinct manner (biased agonism). To shed light on the origin of the divergent activities of the
two endogenous ligands, we performed a conformational study on URP by solution NMR in sodium dodecyl sulfate micelle solution
and compared the obtained NMR structure of URP with that of hU-II previously determined. Finally, we undertook docking
studies between URP, hU-II, and an UT receptor model. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: urotensin-II; urotensin II-related peptide; therapeutic peptide; biased agonism; conformation by NMR; docking studies
in multiple pathophysiological effects interesting primarily cardio-
vascular conditions. Hence, modulation of urotensinergic system
Introduction
is therapeutically appealing in order to deal with different patho-
logical disorders [16,17].
Among a series of regulatory neuropeptides isolated from
urophysis of goby fish, urotensin II (U-II) was characterized as an im-
portant vasoactive cyclic peptide [1]. It has been postulated that
this peptide hormone exerts wide range of pathophysiological
*
Correspondence to: Alfonso Carotenuto, Dipartimento di Farmacia., Università di
Napoli ‘Federico II’, Via D. Montesano, 49, 80131 Naples, Italy. E-mail: alfonso.
carotenuto@unina.it
actions not exclusively in the cardiovascular system. From the iden-
tification of U-II precursor in frog brain [2], it was demonstrated that
DNA encoding for U-II existed in diverse species. Indeed, homolo-
gous peptides of U-II are expressed in non-mammalian and mam-
malian vertebrates, including humans [3–6]. The human U-II
(hU-II) acts as the innate ligand of a G protein-coupled receptor,
more recently known as UT receptor (UTR) [7]. In 2003, Sugo et al.
[8] proved the existence of a paralogue of hU-II so called urotensin
II-related peptide (URP). This peptide is structurally related to hU-II
and displays elevated binding affinity for the human UTR in mam-
mals. Despite of the highly variable N-terminus region sequence
across species (Figure 1), every U-II and URP isopeptide shares the
fully conserved cyclic C-terminal hexapeptide core sequence, c
[Cys-Phe-Trp-Lys-Tyr-Cys], which is responsible of the biological
activity [9]. The genes expressing hU-II and URP are mainly located
in motoneurons of brainstem nuclei and ventral horn in the spinal
cord [10–13]. hU-II and URP messenger RNAs have also been found
in different peripheral tissues such as heart, thymus, pancreas, kid-
ney, intestine, adrenal gland, prostate, and more [3,8,14]. As well as
UTR is broadly distributed in the cardiovascular and central nervous
system and in other peripheral organs and tissues, including kid-
neys, bladder, pancreas, and adrenal gland [15]. This extensive ex-
pression has resulted in the involvement of urotensinergic system
‡
These authors equally contributed as co-first to the paper.
** Special issue of contributions presented at the 14th Naples Workshop on Bioactive
Peptides “The renaissance era of peptides in drug discovery”, June 12–14, 2014, Naples”.
a Department of Pharmacy, University of Naples ‘Federico II’, I-80131, Naples, Italy
b Department of Pharmacy, University of Salerno, I-84084, Fisciano, Salerno, Italy
c
CIRPEB: Centro Interuniversitario di Ricerca sui Peptidi Bioattivi University of
Naples ‘Federico II’, DFM-Scarl, Institute of Biostructures and Bioimaging – CNR,
80134, Naples, Italy
Abbreviations: 1D, 2D, and 3D, one-dimensional, two-dimensional, and three-
dimensional; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DMSO,
dimethylsulfoxide; DQF-COSY, double quantum filtered correlated spectroscopy;
HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexa-fluorophos-
phate; HOBt, 1-hydroxybenzotriazole; MD, molecular dynamics; NOESY, nuclear
Overhauser enhancement spectroscopy; NOE, nuclear Overhauser effect; NMR, nu-
clear magnetic resonance; SDS, sodium dodecyl sulfate; SAR, structure activity re-
lationship; TFA, trifluoroacetic acid; TOCSY, total correlated spectroscopy; Trt,
triphenylmethyl; TSP, 3-(trimethylsilanyl)propionic acid; URP, urotensin II-related
peptide; UTR, urotensin II receptor; h-UTR, human urotensin II receptor; U-II,
urotensin II peptide; hU-II, human urotensin II peptide.
J. Pept. Sci. 2015
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

BRANCACCIO ET AL.
Figure 1. Comparison of the primary structures of mammalian U-II and URP. Conserved amino acids in U-II and URP isoforms are coloured blue (intracyclic
residues) and green (hydrophobic residue). Amino acids adjacent to the cyclic core are coloured red for U-II isoforms (acidic residues) or orange for URP
isoforms (hydrophobic amino acid) to highlight their different physicochemical properties. <Gln represents pyroglutamic acid.
As recently reported, hU-II and URP can exert common as well as
different effects on transcriptional activity, cell proliferation, and
myocardial contractile activities supporting the idea that hU-II and
URP interact with UTR in a distinct manner, i.e. by selecting a spe-
cific UT conformation (biased agonism) [18]. The concept of biased
agonism has recently emerged from various studies, which have
highlighted the notion that specific ligand-induced conformational
changes can lead to particular signalling [19]. Biased agonism
would require specific pockets/interactions within UT receptor,
aimed to select distinct UTR conformations that can discriminate
hU-II and URP biological activities. It has been postulated that a dif-
ferent conformation of the cyclic portion of these two peptides,
β-turn in hU-II versus γ-turn in URP, would cause the selection of dif-
ferent UTR active states, ultimately triggering a slightly different
subset of signalling pathways [18].
(4×), and the deprotection protocol was repeated after each cou-
pling step. The N-terminal Fmoc group was removed as described
in the preceding text, and the peptide was released from the resin
with TFA/Et3SiH/H2O (90 : 5 : 5) for 3 h. The resin was removed by fil-
tration, and the crude peptide was recovered by precipitation with
cold anhydrous ethyl ether to give a white powder that was purified
by RP-HPLC on a semi-preparative C18-bonded silica column
(Vydac 218TP1010, 1.0 × 25 cm) using a gradient of CH3CN in 0.1%
aqueous TFA (from 10 to 90% in 45 min) at a flow rate of 5.0ml/
min. The product was obtained by lyophilization of the appropriate
fractions after removal of the CH3CN by rotary evaporation. Analyt-
ical RP-HPLC indicated purity >98% and molecular weights were
confirmed by ESI-MS analyses performed by API 2000 (Supporting
Information, Table S1).
To investigate the origin of the divergent activities of the two en-
dogenous ligands, we performed conformational studies on URP by
solution NMR and compared the obtained NMR structure of URP
with that of hU-II previously determined. Finally, we undertook
docking studies between URP, hU-II, and a newly developed UT
receptor model.
General Method of Oxidation and Cyclization
Peptide was oxidized by the syringe pump method previously
reported [20]. The linear peptide (300–500 mg) was dissolved
in 40 ml of 50% H2O/25% acetonitrile/25% methanol, and nitro-
gen gas was passed through the solution for 20 min. Five
millilitres of saturated ammonium acetate solution were added,
and the pH was taken to 8.5 with NH4OH. The peptide solution
was then added at room temperature via syringe pump to a
stirred oxidant solution. The oxidant solution was prepared as
follows: 2 equiv of potassium ferricyanide were dissolved in
400 ml of H2O/200 ml of acetonitrile/200 ml of methanol. To this
solution was added 100 ml of saturated ammonium acetate, and
the pH was then taken to 8.5 with NH4OH. The peptide solution
was added at such a rate that approximately 10 mg of peptide
were delivered per hour per litre of the oxidant. After the addi-
tion of peptide was complete, the reaction mixture was stirred
for an additional 5–6 h and then taken to pH 3.5 with glacial
acetic acid. Amberlite IRA-68 (Cl – form) was added to remove
the iron ions, and the solution stirred for 20 min and then fil-
tered. The solution was concentrated using a rotary evaporator
at 30°C and then lyophilized. The material thus obtained was
dissolved in glacial acetic acid, filtered to remove inorganic salts,
and relyophilized. The crude cyclic peptide was purified by pre-
parative HPLC on the system described in the preceding texts,
using a gradient of 100% buffer for 20 min, then 0–20% acetoni-
trile in 5 min, followed by 20–60% acetonitrile in 40 min, all at
40 ml/min. Again, the peptide eluted near 50% organic/50%
buffer. The purity of the cyclic peptide was checked by analytical
HPLC (C-18 column, Vydac 218TP104, 4,6× 25 cm), using a
Shimadzu SPD 10A vp with detection at 230 and 254 nm and
by TLC in four solvent systems in silica gel with detection by
Materials and Methods
Peptide Synthesis
Nα-Fmoc-protected amino acids, HBTU, and HOBt were purchased
from Inbios (Naples, Italy). Wang resin was purchased from Ad-
vanced ChemTech (Louisville, KY). Peptide synthesis solvents, re-
agents, as well as CH3CN for HPLC were reagent grade and were
acquired from commercial sources and used without further purifi-
cation unless otherwise noted. The synthesis of URP was performed
in a stepwise fashion via the solid-phase method. Nα-Fmoc-Val-OH
was coupled to Wang resin in the presence of HBTU (3 equiv), HOBt
(3 equiv), DIEA (6 equiv) and DMAP in catalytic amount, to facilitate
ester formation, in DMF for 3 h at rt. The following protected amino
acids were then added stepwise: Nα-Fmoc-Cys(Trt)-OH, Nα-Fmoc-
Tyr(tBu)-OH, Nα-Fmoc-Lys(Nε-Boc)-OH, Nα-Fmoc-Trp(Nin-Boc)-OH,
Nα-Fmoc-Phe-OH, Nα-Fmoc-Cys(Trt)-OH, and Nα-Fmoc-Ala-OH. Each
coupling reaction was accomplished using a threefold excess of
amino acid with HBTU and HOBt in the presence of DIEA (6 equiv).
The Nα-Fmoc protecting groups were removed by treating the
protected peptide resin with a 25% solution of piperidine in DMF,
(1× 5 and 1 × 20 min). The peptide resin was washed three times
with DMF and the subsequent coupling step was initiated in a step-
wise manner. All reactions were performed under a N2 atmosphere.
The peptide resin was washed with DCM (3×), DMF (3×), and DCM
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015

URP AND hU-II CONFORMATION AND INTERACTION WITH UT RECEPTOR
UV light, iodine vapours, and ninhydrin. The analytical data of
URP are given in the Supporting Information (Table S1).
h-UTR Model and Docking
3D structure predictions of h-UTR were generated by I-TASSER
server for protein structure and function prediction, which is based
on a threading alignment algorithm [32–34]. Five models of h-UTR
were obtained using I-TASSER servers. The best scored model
(model 1, refer to Results Section) was used for docking studies.
The initial poses for the h-UTR–URP complex are generated by
docking the lowest energy conformers of URP obtained by NMR
to the h-UTR model using the program HADDOCK 2.0 [35,36]. For
comparative purpose, also the best scored NMR structure of hU-II
[30] was docked to the same h-UTR model. Only the distance be-
tween Nε of Lys8 and carboxyl oxygens of Asp130 on TM3 was used
as a restraint (2.7 1 Å) in the docking studies because the last res-
idue is generally regarded as the ligand recognition site [37].
Considering the receptor, only the side chains of residues en-
gaged in orthosteric ligand/GPCR binding in the complexes used
as templates by I-TASSER (PDB IDs: 4n6h, 4dkl, 4buo; refer to Results
Section) were considered flexible in the docking procedure (active
residue: W116, L126, F127, D130, F131, M134, V184, M188, H208,
L212, F274, W275, W277, Q278, Y305). Instead, all peptide’s atoms
were held frozen (passive residue).
Materials for NMR
2
99.9% H2O were obtained from Aldrich (Milwaukee, USA); 98%
sodium dodecyl sulfate (SDS)-d25 was obtained from Cambridge
Isotope Laboratories, Inc. (Andover, USA), [(2,2,3,3-tetradeuterio-
3-(trimethylsilanyl)]propionic acid (TSP) from MSD Isotopes
(Montreal, Canada).
NMR Spectroscopy
The samples for NMR spectroscopy were prepared by dissolving
the appropriate amount of URP in 0.45 ml of 1H2O (pH 5.5),
0.05ml of 2H2O to obtain a concentration 1–2 mM of peptide. For
the sample in micelle solution, SDS-d25 was also added to a concen-
tration of 200mM. NMR spectra were recorded on a Varian INOVA
700 MHz spectrometer equipped with a z-gradient 5 mm triple-
resonance probe head. Spectra in water solution and micelle were
recorded at a temperature of 10 and 25°C, respectively. The spectra
were calibrated relative to TSP (0.00ppm) as internal standard. 1D
NMR spectra were recorded in the Fourier mode with quadrature
detection. The water signal was suppressed by gradient echo [21].
2D DQF-COSY [22,23], TOCSY [24], and NOESY [25] spectra were re-
corded in the phase-sensitive mode using the method from USA
[26]. Data block sizes were 2048 addresses in t2 and 512 equidistant
t1 values. Before Fourier transformation, the time domain data ma-
trices were multiplied by shifted sin2 functions in both dimensions.
A mixing time of 70 ms was used for the TOCSY experiments.
NOESY experiments were run with mixing times in the range of
150–300 ms. The qualitative and quantitative analyses of DQF-
COSY, TOCSY, and NOESY spectra were obtained using the interac-
tive program package XEASY [27]. 3JHN-Hα coupling constants were
obtained from 1D 1H NMR and 2D DQF-COSY spectra. The temper-
ature coefficients of the amide proton chemical shifts were calcu-
Refinement of each pose was achieved by in vacuo energy min-
imization with the Discover algorithm using the steepest descent
and conjugate gradient methods until a RMSD of 0.05 kcal/mol/Å
was reached. The backbone atoms of the TM and IL domains of
the h-UTR were held in their position; the ligand and extracellular
loops (ELs) were free to relax. Molecular graphics images of the
complexes were produced using the UCSF Chimera package [31].
Results
Chemistry
The peptide URP was synthesized by solid-phase strategy. The
Fmoc/tBu orthogonal protecting groups were used, and the syn-
thesis was accomplished in a manual reaction vessel [38] (Exper-
imental Section). The crude peptide was purified by using a
semi-preparative RP-HPLC equipped with C-18 bonded silica col-
umn (Vydac 218TP1010). The purified peptide was analysed by
analytical RP-HPLC showing >98% purity. Exact molecular weight
of the peptide was proved by mass spectrometry and amino
acid analysis (Supporting Information, Table S1).
1
lated from 1D H NMR and 2D TOCSY experiments performed at
different temperatures in the range 25–40°C by means of linear
regression.
Structural Determinations
The NOE-based distance restraints were obtained from NOESY
spectra collected with a mixing time of 200 ms. The NOE cross
peaks were integrated with the XEASY program and were con-
verted into upper distance bounds using the CALIBA program in-
corporated into the program package DYANA [28]. Cross peaks
that were overlapped more than 50% were treated as weak
restraints in the DYANA calculation. Only NOE-derived con-
straints (Supporting Information) were considered in the anneal-
ing procedures. An ensemble of 200 structures was generated
with the simulated annealing of the program DYANA. An error-
tolerant target function (tf-type = 3) was used to account for
the peptide intrinsic flexibility. From the produced 200 confor-
mations, 50 structures were chosen, whose interproton distances
best fitted NOE derived distances and then refined through suc-
cessive steps of restrained and unrestrained energy minimization
using the Discover algorithm (Accelrys, San Diego, CA) and the
consistent valence force field [29] as previously described [30].
The final structures were analysed using the InsightII program
(Accelrys, San Diego, CA). Molecular graphics images of the com-
plexes were produced using the UCSF Chimera package [31].
NMR Analysis
1D and 2D NMR spectra were collected in water and 200 mM aque-
ous solution of SDS for URP. Micelle solution was used because we
have studied the NMR structure of hU-II and other UTR agonists
[30,39,40] and antagonist [41] in this medium.
Complete 1H NMR chemical shifts assignment was accomplished
according to the Wüthrich procedure [42] via the analysis of DQF-
COSY [22,23], TOCSY [24], and NOESY [25] spectra using the XEASY
software package (Supporting Information, Tables S2 and S3) [27].
Considering the spectra in water solution, many NMR parameters
indicate structural flexibility (Table S2). For example, Hα chemical
shift values are all close (Δδ < 0.1ppm) to the corresponding ones
in random coil peptides [43] apart that of Lys8 (for easy comparison,
peptide numbering of URP follows that of hU-II).
In contrast, NMR parameters derived from spectra acquired in
SDS micelles are typical of a structured peptide (Table S3). Fur-
thermore, such parameters indicated that URP structure is similar
J. Pept. Sci. 2015
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

BRANCACCIO ET AL.
to other UTR ligands previously studied by us, especially to its
paralog hU-II. For comparison purpose, Hα chemical shifts of resi-
dues of hU-II constituting the cyclic moiety common to URP are
also reported in Table S3. In particular, NOE contacts between
Hα–NHi+2 of Trp7 and Tyr9 and between NH–NHi+1 of Lys8 and
Tyr9 indicated the presence of a β-turn. The observation of slowly
exchanging NH resonance of residue 9, and low value of the
temperature coefficient for this proton (ÀΔδ/ΔT < 3.0ppb/K) con-
firmed this result. A number of long-range NOEs including Hα–NH
connectivities between residues 5, 11, and 10, 6 and a NH–NH
connectivity between residues 6 and 9 supported the existence
of a short stretch of antiparallel β-sheet involving residues 5–6
3
and 10–11. Also, large values of JHN-Hα coupling constants
(3JHN-Hα > 8.0Hz) for residues 5 and 9–11 confirm a β-sheet struc-
ture (Table S3). Overall data supported the existence of the
β-hairpin structure in URP. Furthermore, many NOE interactions
between Trp7 with Lys8 side chains implied that those side chains
are close. Also, Tyr9 side chain shows NOE contacts with Lys8,
while Phe6 shows contacts with Val11.
Constraints derived from NMR data were used as the input for a
structure calculation by simulated annealing. Structure calculations
using NMR data from URP spectra acquired in water gave not
converging results (backbone RMSD> 2 Å for the 10 lowest energy
conformers; data not shown). Differently, using the NMR constraints
from SDS micelle solution (Table S4), an ensemble of well-defined
structures could be obtained. In fact, the 10 lowest energy struc-
tures (Figure 2) showed a backbone RMSD of 0.34 Å and satisfied
the NMR-derived constraints (violations smaller than 0.20 Å). As
shown, URP folds into a type II′ β-hairpin structure along residues
5–10. Considering the side chain orientation, Phe6, Trp7, Lys8, and
Tyr9 χ1 angles showed a high preference for gÀ, trans, g+, and gÀ
rotamers, respectively. URP-obtained NMR structure is very similar
to those of other UTR ligands previously found by us and, in partic-
ular, to that of hU-II (Figure 3).
Figure 3. Stereoview of the superposition of the lowest energy conformer
of URP (coulor code as in Figure 2) and hU-II (carbon atoms in grey).
Structures were superimposed using the backbone heavy atoms of
residues 5–10.
The top template used is the high-resolution crystal structure of hu-
man δ-opioid receptor (h-DOR, PDB ID: 4n6h) [44]. Other templates
used for threading are the crystal structure of mouse μ-opioid
receptor (m-MOR, PDB ID: 4dkl) [45] and the crystal structure of a
neurotensin receptor 1 mutant (NTSR1, PDB ID: 4buo) [46]. The
most important score in I-TASSER models is the confidence score
(C-score), ranging from À5 to +2. The C-score is computed from
the threading alignments for the estimated quality of the models.
A C-score >À1.5 implies a model with a correct fold [34]. Model 1
of h-UTR (C-score = 1.56) with higher C-scores was chosen as the
best model and was considered for the analyses. The model predic-
tions were judged using the template modelling (TM) score and
root mean-squared difference (rmsd). The TM score is a measure
of the structural similarity between the model and the native struc-
ture. A TM score >+0.5 suggests a model with correct topology. The
TM score of model 1 for h-UTR was 0.93 0.06Å. The expected
RMSDs were 3.0 2.2Å. Additionally, to confirm the reliability of
the model 1, the program PROCHECK [47] was employed. All amino
acids in the α-helices were found in the favoured region of the
right-handed α-helix in the Ramachandran plot. There were no cis
peptide bonds, and there were no bump regions in the calculated
h-UTR models. The results reveal that our 3D model for h-UTR is
acceptable and of high quality. Worth to note, the selected model
1 maintain most of the molecular signatures that feature class A
GPCR [48]. For example, in the selected model are present 24 out
of the 24 inter-TM contacts of the consensus network found in
the GPCR structures. A superposition of h-DOR crystal structure
and h-UTR model 1 is shown in Figure S1 (Supporting Information).
Docking procedures using the program HADDOCK [35,36]
clustered 198 structures in two clusters for both the complexes
h-UTR/URP and h-UTR/hU-II. Statistics and energy terms are
reported in Table 1. Best scored complexes of h-UTR/URP and
h-UTR/hU-II are shown in Figures 4 and 5, respectively.
h-UTR Model and Docking
3D models of h-UTR were generated based on the structure of
other GPCR, using the I-TASSER server [32–34]. Five models of
h-UTR were generated using I-TASSER server. I-TASSER output also
contained top ranks of templates used for the structure prediction.
Discussion
Figure 2. Stereoview of the superposition of the 10 lowest energy
conformers of URP. Structures were superimposed using the backbone
heavy atoms of residues 5–10. Heavy atoms are shown with different
colours (carbon, green; nitrogen, blue; oxygen, red; sulfur, yellow).
Hydrogen atoms are not shown for clarity.
U-II and URP could exert common as well as distinct actions on
cell proliferation, transcriptional activity, and myocardial
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015

URP AND hU-II CONFORMATION AND INTERACTION WITH UT RECEPTOR
Table 1. Statistics and energy terms of the calculated complexes.
Complex
Cluster n.
H scorea
Cluster size
RMSDb
VdWc
Eletrd
Desolve
h-UTR/URP
h-UTR/URP
h-UTR/hU-II
h-UTR/hU-II
1
2
1
2
À130.1 2.6
À102.1 2.5
À144.2 2.5
À113.4 13.2
191
7
0.5 0.3
0.8 0.0
1.3 0.0
1.1 0.0
À56.6 3.9
À50.7 2.8
À56.2 6.5
À56.6 4.0
À150.9 21.8
À166.3 17.7
À230.2 26.2
À210.2 10.4
À49.3 3.5
À28.4 7.5
À48.5 2.5
À24.8 9.8
193
5
aHADDOCK score.
bRMSD from the overall lowest energy structure.
cVan der Waals energy.
dElectrostatic energy.
eDesolvation energy. All terms are given in Kcal/mol.
Figure 4. (a) Stereoview of h-UTR model complexed with URP. URP heavy atoms are colour coded as in Figure 2. Receptor backbones are represented in
azure and labelled. (b) Stereoview of URP within the binding pocket of h-UTR. Hydrogen bonds are represented with dashed lines. Labels of UTR residues
involved in previous mutagenesis studies are evidenced in red. For the sake of clarity here and throughout the manuscript, the residue numbers of the
ligands are reported as apex while those of the receptor are not.
contractile activities supporting the idea that U-II and URP inter-
act with UTR in a distinct manner, i.e. selecting a specific UTR
conformation (biased agonism) [18]. Biased agonism would re-
quire specific pockets/interactions within UTR, finalized to select
distinct UTR conformations that may discriminate U-II and URP
biological activities. It was hypothesized that a different confor-
mation of the cyclic portion of the two peptides, β-turn in U-II
versus γ-turn in URP, would cause the selection of different
UTR active states, ultimately triggering a slightly different subset
of signalling pathways [16,18]. Hence, we first performed a
conformational study on URP by solution NMR. NMR study
was performed both in water and in SDS micelle solution.
SDS micelles were used because they mimic the cell mem-
brane where UTR is located. A membrane-mediated mechanism
of interaction between peptides and their receptors has been
postulated [49,50]. According to that mechanism, cell membrane
would facilitate peptide increase of local concentration, reduction
of rotational and translational freedom, and folding. In fact, several
J. Pept. Sci. 2015
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

BRANCACCIO ET AL.
Figure 5. (a) Stereoview of h-UTR model complexed with hU-II. hU-II heavy atoms are colour coded as in Figure 2, apart carbon atoms that are in orange.
Receptor backbones are represented in azure and labelled. (b) Stereoview of hU-II within the binding pocket of h-UTR. Hydrogen bonds are represented
with dashed lines. Labels of UTR residues involved in previous mutagenesis studies are evidenced in red.
conformational studies on peptides have been performed in
micelle solution [50–54]. Notably, a clear correlation between the
conformation in SDS micelles of hU-II analogues and their biologi-
cal activity has been found by us [40,55,56]. NMR-derived structures
of URP in SDS solution are shown in Figure 2. Clearly, β-hairpin
conformation of the backbone and the side chain cluster of the
pharmacophoric residues Trp7, Lys8, and Tyr9, which are distinctive
of UTR peptide agonists, are still observable in URP structure.
Whereby, URP-obtained NMR structure is very similar to those
of other UTR ligands previously found by us and, in particular,
to that of hU-II (Figure 3). It comes out that divergent actions
of URP and hU-II cannot derive from different conformation of
the ligands as hypothesized by Chatenet et al. [18]. That hypothesis
was inspired by the comparison of the NMR structure of hU-II
obtained by us in SDS micelle solution [30] and NMR structure
of URP obtained by Chatenet et al. in water solution [9].
The last structure is characterized by an inverse γ-turn centred
on the Trp-Lys-Tyr sequence as opposite to the β-turn hosted on
the corresponding residues of hU-II. An inverse γ-turn centred on
the Trp-Lys-Tyr is compatible with the data obtained by us in water
solution, namely with a NOE contact between Hα–NHi+2 of Trp7 and
Tyr9. However, structure calculations using NMR data from URP
spectra acquired in water gave not converging results (data
not shown) probably because of a high structural flexibility of the
peptide in plain buffer.
In order to determine whether the divergent actions of URP
and hU-II derive from different interaction with the receptor,
NMR-derived structures of URP and hU-II were docked within a
model of h-UTR built by homology using the I-TASSER online
server [32–34]. We have already built an h-UTR model [39]. It
was based on the rhodopsin crystal structure [57]. The current
model is based mainly on the h-DOR crystal structure [44].
Because the last is a diffusible ligand (peptide) binding GPCR,
which share higher sequence homology with h-UTR (TM
sequence identities: 34.5% to h-DOR vs 21.8% to Rho), hence it
can be considered a more reliable model than the previous
one. In the docking procedures, we hypothesized that peptides
bind in the extracellular side of the TM bundle, as observed
for all the ligands of class A GPCR [48] and confirmed by muta-
genesis studies [58–60]. In this context, only the side chains of
residues engaged in orthosteric ligand/GPCR binding in com-
plexes with known crystal structure were considered flexible in
the docking procedure. In particular, we took in account
h-DOR/naltrindole (PDB ID: 4n6h), m-MOR/β-funaltrexamine
(PDB ID: 4dkl), and NTSR1/neurotensin (PDB ID: 4buo) complexes
because they were used as templates by the unbiased homol-
ogy building procedure of I-TASSER (refer to the preceding
texts). A superposition of the putative binding site of h-UTR
model and h-DOR/naltrindole complex is shown in Figure S2
(Supporting Information) as an example.
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015

URP AND hU-II CONFORMATION AND INTERACTION WITH UT RECEPTOR
3 Beauvillain JC, Conlon JM, Bern HA, Vaudry H. Cloning of the cDNA
encoding the urotensin II precursor in frog and human reveals intense
expression of the urotensin II gene in motoneurons of the spinal cord.
Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15803–15808.
4 Coulouarn Y, Jegou S, Tostivint H, Vaudry H, Lihrmann I. Cloning,
sequence analysis and tissue distribution of the mouse and rat
urotensin II precursors. FEBS Lett. 1999; 457: 28–32.
Best poses of complexes of h-UTR/URP and h-UTR/hU-II are
shown in Figures 4 and 5, respectively. For both peptides, the pre-
dicted binding site is located among TM3/TM7, EL2, and EL3. The
β-hairpin is aligned with the receptor helical axis, with the N-
termina and C-termina pointing towards the extracellular side. Main
interactions between the peptides and UTR are shown in Figure 4b
for h-UTR/URP and 5b for h-UTR/hU-II. These findings are in accor-
dance with previous mutagenesis results [58–60]. In fact, many of
the receptor residues, involved in the peptides binding in our
model, face the binding site pocket of the UT receptor as demon-
strated using the substituted-cysteine accessibility method. For
some others, the replacement with a cysteine residue caused a
complete loss of affinity for the ligands. All these residues are evi-
denced in Figures 4b–5b. Furthermore, both hU-II and URP interact
with the ELs but EL1 in accordance with experimental data [61].
Interestingly, comparing our complex model with the peptide-
bound GPCR crystal structures solved to date (NTSR1/neurotensin
[46] and CXCR4/CVX15 [62]), it can be argued that hU-II (and URP,
data not shown) binds to the h-UTR in a similar fashion as
neurotensin to NTSR1 and CVX15 to CXCR4 (Figure S3, Supporting
Information).
5 Mori M, Sugo T, Abe M, Shimomura Y, Kurihara M, Kitada C, Kikuchi K,
Shintani Y, Kurokawa T, Onda H, Nishimura O, Fujino M. Urotensin II is
the endogenous ligand of a G-protein coupled orphan receptor, SENR
(GPR14). Biochem. Biophys. Res. Commun. 1999; 265: 123–129.
6 Elshourbagy NA, Douglas SA, Shabon U, Harrison S, Duddy G, Sechler JL,
Ao Z, Maleef BE, Naselsky D, Disa J, Aiyar NV. Molecular and
pharmacological characterization of genes encoding urotensin II-
related peptides and their cognate G-protein coupled receptors from
the mouse and monkey. Br. J. Pharmacol. 2002; 136: 9–22.
7 Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar RV, Romanic AM,
Louden CS, Foley JJ, Sauermelch CF, Coatney RW, Ao Z, Disa J,
Holmes SD, Stadel JM, Martin JD, Liu WS, Glover GI, Wilson S, McNutty DE,
Ellis CE, Eishourbagy NA, Shabon U, Trill JJ, Hay DVP, Ohlstein EH,
Bergsma DJ, Douglas SA. Human urotensin-II is a potent vasoconstrictor
and agonist for the orphan receptor GPR14. Nature 1999; 401: 282–286.
8 SugoT, MurakamiY,ShimomuraY,HaradaM, AbeM, IshibashiY,KitadaC,
Miyajima N, Suzuki N, Mori M, Fujino M. Identification of urotensin II-
related peptide as the urotensin II immunoreactive molecule in the rat
brain. Biochem. Biophys. Res. Commun. 2003; 310: 860–868.
While the two peptides hU-II and URP share similar interactions
with the receptor concerning the cyclic region, N-terminal region
of hU-II establishes large interactions with extracellular loops EL2
of h-UTR. In particular, charge-reinforced hydrogen bonds between
Glu1 and Lys196 and between Asp4 and Arg193 are observable in
hU-II/h-UTR complex (Figure 5). Those interactions cannot be pres-
ent in URP/h-UTR complex. In agreement with our model, dissocia-
tion kinetics experiments revealed a putative interaction between
UTR and the glutamic residue at position 1 of hU-II. Indeed, it was
observed that the replacement of this residue by an alanine, i.e.
[Ala1]hU-II, caused an increase in the dissociation rate of hU-II but
not URP [18].
9 Chatenet D, Dubessy C, Leprince J, Boularan C, Carlier L,
Segalas-Milazzo I, Guilhaudis L, Oulyadi H, Davoust D, Scalbert E,
Pfeiffer B, Renard P, Tonon MC, Lihrmann I, Pacaud P, Vaudry H.
Structure-activity relationships and structural conformation of a novel
urotensin II-related peptide. Peptides 2004; 25: 1819–1830.
10 Chartrel N, Conlon JM, Collin F, Braun B, Waugh D, Vallarino M,
Lahrichi SL, Rivier JE, Vaudry H. Urotensin II in the central nervous
system of the frog Rana ridibunda: immunohistochemical localization
and biochemical characterization. J. Comput. Neurol. 1996; 364: 324–339.
11 Coulouarn Y, Fernex C, Jegou S, Henderson CE, Vaudry H, Lihrmann I
Specific expression of the urotensin II gene in sacral motoneurons of
developing rat spinal cord. Mech. Dev. 2001; 101: 187–190.
12 Pelletier G, Lihrmann I, Vaudry H. Role of androgens in the regulation of
urotensin II precursor mRNA expression in the rat brainstem and spinal
cord. Neuroscience 2002; 115: 525–532.
13 Pelletier G, Lihrmann I, Dubessy C, Luu-The V, Vaudry H, Labrie F.
Androgenic down-regulation of urotensin II precursor, urotensin II-
related peptide precursor and androgen receptor mRNA in the mouse
spinal cord. Neuroscience 2005; 132: 689–696.
14 Novellino E, Grieco P, Caraglia M, Budillon A, Franco R, Addeo SR.
Peptidic and non peptidic ligands for immunodetection of urotensin-II
receptor. PCT Int. Appl. 2008; 21pp. WO 2008095995.
15 Vaudry H, Do Rego JC, Le Mevel JC, Chatenet D, Tostivint H, Fournier A.
Urotensin II, from fish to human. Ann. N.Y. Acad. Sci. 2010; 1200: 53–66.
16 Chatenet D, Nguyen TT, Létourneau M, Fournier A. Update on the
urotensinergic system: new trends in receptor localization, activation,
and drug design. Front. Endocrinol. 2013; 3: 1–13.
Conclusions
We demonstrated that distinct pathophysiological roles for URP
and hU-II are not related to different conformations of the two pep-
tides, but they likely arise from their different interactions with the
UT receptor. Those interactions can stabilize different active confor-
mations of UTR that, in turn, can select specific subset of secondary
messengers depending on the ligand-induced adopted
conformation.
17 Maryanoff EB, Kinney AW. Urotensin-II receptor modulators as potential
drugs. J. Med. Chem. 2010; 53: 2695–2708.
18 Chatenet D, Létourneau M, Nguyen QT, Doan ND, Dupuis J, Fournier A.
Discovery of new antagonists aimed at discriminating UII and URP-
mediated biological activities: insight into UII and URP receptor
activation. Br. J. Pharmacol. 2013; 168: 807–821.
19 Patel CB, Noor N, Rockman HA. Functional selectivity in adrenergic and
angiotensin signaling systems. Mol. Pharmacol. 2010; 78: 983–992.
20 Misika A, Hruby VJ. Optimization of disulfide bond formation. Pol. J.
Chem. 1994; 68: 893–899.
Acknowledgement
Part of this work was supported by Italian Ministry of Education,
University and Research (PRIN 2008, 2008TSWB8K_002; PRIN
2009, 2009EL5WBP_004) and a grant from ‘Regione Campania’ –
Laboratori Pubblici progetto ‘Hauteville’.
21 Hwang TL, Shaka AJ. Water suppression that works. Excitation sculpting
using arbitrary wave-forms and pulsed-field gradients. J. Magn. Res.
1995; 112: 275–279.
22 Piantini U, Sorensen OW Ernst RR Multiple quantum filters for elucidating
NMR coupling network. J. Am. Chem. Soc. 1982; 104: 6800–6801.
23 Marion D, Wüthrich K. Application of phase sensitive two-dimensional
correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin
coupling constants in proteins. Biochem. Biophys. Res. Commun. 1983;
113: 967–974.
References
1 Pearson D, Shively JE, Clark BR, Geschwind II, Barkley M, Nishioka RS,
Bern HA. Urotensin II: a somatostain-like peptide in the caudal
neurosecretory system of fishes. Proc. Natl. Acad. Sci. U. S. A. 1980; 77:
5021–5024.
2 Conlon JM, O’Harte F, Smith DD, Tonon MC, Vaudry H. Isolation and
primary structure of urotensin II from the brain of a tetrapod, the frog
Rana ridibunda. Biochem. Biophys. Res. Commun. 1992; 188: 578–583.
24 Braunschweiler L, Ernst RR. Coherence transfer by isotropic mixing:
application to proton correlation spectroscopy. J. Magn. Reson. 1983;
53: 521–528.
J. Pept. Sci. 2015
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

BRANCACCIO ET AL.
25 Jenner J, Meyer BH, Bachman P, Ernst RR. Investigation of exchange
processes by two-dimensional NMR spectroscopy. J. Chem. Phys. 1979;
71: 4546–4553.
47 Laskowski RA, Mac Arthur MW, Moss DS, Thornton JM. PROCHECK: a
program to check the stereochemical quality of protein structures.
J. Appl. Crystallogr. 1993; 26: 283–291.
26 States DJ, Haberkorn RA, Ruben DJ. A two-dimensional nuclear
Overhauser experiment with pure absorption phase in four quadrants.
J. Magn. Reson. 1982; 48: 286–292.
48 Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF Babu MM.
Molecular signatures of G-protein-coupled receptors. Nature 2013; 494:
185–94.
27 Bartels C, Xia T, Billeter M, Güntert P Wüthrich K The program XEASY for
computer-supported NMR spectral analysis of biological
macromolecules. J. Biomol. NMR 1995; 6: 1–10.
28 Güntert P, Mumenthaler C Wüthrich K Torsion angle dynamics for NMR
structure calculation with the new program DYANA. J. Mol. Biol. 1997;
273: 283–298.
29 Maple J, Dinur U, Hagler AT. Derivation of force fields for molecular
mechanics and dynamics from ab initio energy surface. Proc. Natl.
Acad. Sci. U. S. A. 1988; 85: 5350–53.
49 Moroder L, Romano R, Guba W, Mierke DF, Kessler H, Delporte C,
Winand J, Christophe J. New evidence for a membrane bound
pathway in hormone receptor binding. Biochemistry 1993; 32:
13551–13559.
50 Grieco P, Giusti L, Carotenuto A, Campiglia P, Calderone V, Lama T,
Gomez-Monterrey I, Tartaro G, Mazzoni MR, Novellino E. Morphiceptin
analogues containing a dipeptide mimetic structure: an investigation
on the bioactive topology at the μ-receptor. J. Med. Chem. 2005; 48:
3153–3163.
30 CarotenutoA, GriecoP,CampigliaP,NovellinoE, RoveroP.Unravelingthe
active conformation of urotensin II. J. Med. Chem. 2004; 47: 1652–1661.
31 Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM,
Meng EC, Ferrin TE. UCSF Chimera—a visualization system for
exploratory research and analysis. J. Comput. Chem. 2004; 25: 1605–1612.
32 Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinf
2008; 9: 40. DOI: 10.1186/1471-2105-9-40.
33 Kelley LA. Sternberg MJ Protein structure prediction on the Web: a case
study using the Phyre server. Nat. Protoc. 2009; 4: 363–371.
34 Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated
protein structure and function prediction. Nat. Protoc. 2010; 5: 725–738.
35 de Vries SJ, van Dijk M Bonvin AMJJ The HADDOCK web server for data-
driven biomolecular docking. Nat. Protoc. 2010; 5: 883–897.
36 Wassenaar TA, van Dijk M, Loureiro-Ferreira N, van der Schot G,
de Vries SJ, Schmitz C, van der Zwan J, Boelens R, Giachetti A, Ferella L,
Rosato A, Bertini I, Herrmann T, Jonker HRA, Bagaria A, Jaravine V,
Guntert P, Schwalbe H, Vranken WF, Doreleijers JF, Vriend G,
Vuister GW, Franke D, Kikhney A, Svergun DI, Fogh R, Ionides J,
Laue ED, Spronk C, Jurka S, Verlato M, Badoer S, Dal Pra S,
Mazzucato M, Frizziero E, Bonvin AMJJ. WeNMR: structural biology on
the grid. J. Grid. Comp. 2012; 10: 743–767.
51 D’Addona D, Carotenuto A, Novellino E, Piccand V, Reubi JC, Di Cianni A,
Gori F, Anna Papini AM, Ginanneschi M. Novel sst5-selective
somatostatin dicarba-analogues: synthesis and conformation-affinity
relationships. J. Med. Chem. 2008; 51: 512–520.
52 Di Cianni A, Carotenuto A, Brancaccio D, Ettore Novellino E,
Jean Claude Reubi JC, Karin Beetschen K, Anna Maria Papini AM,
Mauro Ginanneschi M. Novel octreotide dicarba-analogues with high
affinity and different selectivity for somatostatin receptors. J. Med.
Chem. 2010; 53: 6188–6197.
53 Grieco P, Brancaccio D, Novellino E, Hruby VJ, Carotenuto A.
Conformational study on cyclic melanocortin ligands and new insight
into their binding mode at the MC4 receptor. Eur. J. Med. Chem. 2011;
46: 3721–3733.
54 Mollica A, Carotenuto A, Novellino E, Limatola A, Costante R,
Pinnen F, Stefanucci A, Pieretti S, Borsodi A, Samavati R, Zador F,
Benyhe S, Davis P, Porreca F, Hruby VJ. Novel cyclic biphalin
analogue with improved antinociceptive properties. ACS Med. Chem.
Lett. 2014; 5: 1032–1036.
55 Carotenuto A, Auriemma L, Merlino F, Limatola A, Campiglia P,
Gomez-Monterrey I, di Villa Bianca R, Brancaccio D, Santicioli P,
Meini S, Maggi CA, Novellino E, Grieco P. New insight into the binding
mode of peptides at urotensin-II receptor by Trp-constrained
analogues of P5U and urantide. J. Pept. Sci. 2013; 19: 293–300.
56 Brancaccio D, Limatola A, Campiglia P, Gomez-Monterrey I, Novellino E,
Grieco P, Carotenuto A. Urantide conformation and interaction with
the urotensin-II receptor. Arch. Pharm. 2014; 347: 185–192.
57 Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA,
Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M.
Crystal structure of rhodopsin: a G protein-coupled receptor. Science
2000; 289: 739–745.
37 Kinney WA, Almond HR, Qi J, Smith CE, Santulli RJ, de Garavilla L,
Andrade-Gordon P, Cho DS, Everson AM, Feinstein MA, Leung PA,
Maryanoff BE. Structure-function analysis of urotensin II and its use in
the construction of a ligand-receptor working model. Angew. Chem.
Int. Ed. 2002; 41: 2940–2944.
38 Stewart JM, Young JD. Solid Phase Peptide Synthesis, Pierce Chemical:
Rockford, IL,1984.
39 Grieco P, Carotenuto A, Campiglia P, Gomez-Monterrey I, Auriemma L,
Sala M, Marcozzi C, d’Emmanuele di Villa Bianca R, Brancaccio D,
Rovero P, Santicioli P, Meini S, Maggi CA, Novellino E. New insight into
the binding mode of peptide ligands at urotensin-II receptor:
structure-activity relationships study on P5U and urantide. J. Med.
Chem. 2009; 52: 3927–3940.
58 Holleran BJ, Domazet I, Beaulieu ME, Yan LP, Guillemette G, Lavigne P,
Escher E, Leduc R. Identification of transmembrane domain 6 & 7
residues that contribute to the binding pocket of the urotensin II
receptor. Biochem. Pharmacol. 2009; 77: 1374–1382.
40 Carotenuto A, Auriemma L, Merlino F, Yousif AM, Marasco D, Limatola A,
Campiglia P, Gomez-Monterrey I, Santicioli P, Meini S, Maggi CA,
Novellino E. Grieco P. Lead optimization of P5U and urantide:
discovery of novel potent ligands at the urotensin-II receptor. J. Med.
Chem. 2014; 57: 5965–5974.
59 Sainsily X, Cabana J, Boulais PE, Holleran BJ, Escher E, Lavigne P, Leduc R.
Identification of transmembrane domain 3, 4 & 5 residues that
contribute to the formation of the ligand-binding pocket of the
urotensin-II receptor. Biochem. Pharmacol. 2013; 86: 1584–1593.
60 Sainsily X, Cabana J, Holleran BJ, Escher E, Lavigne P, Leduc R.
41 Grieco P, Carotenuto A, Campiglia P, Marinelli L, Lama T, Patacchini R,
Santicioli P, Maggi CA, Rovero P, Novellino E. Urotensin-II receptor
ligands. From agonist to antagonist activity. J. Med. Chem. 2005; 48:
7290–7297.
Identification of transmembrane domain 1 & 2 residues that
contribute to the formation of the ligand-binding pocket of the
urotensin-II receptor. Biochem. Pharmacol. 2014. DOI: 10.1016/j.
bcp.2014.08.023.
42 Wüthrich K. NMR of Proteins and Nucleic Acids, John Wiley & Sons, Inc:
New York,1986.
43 Wishart DS, Sykes BD, Richards FM. The chemical shift index: a fast
method for the assignment of protein secondary structure through
NMR spectroscopy. Biochemistry 1992; 31: 1647–1651.
44 Fenalti G, Giguere PM, Katritch V, Huang XP, Thompson AA, Cherezov V,
Roth BL, Stevens RC. Molecular control of δ-opioid receptor signalling.
Nature 2014; 506: 191–196.
45 Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK,
Pardo L, Weis WI, Kobilka BK, Granier S. Crystal structure of the
μ-opioid receptor bound to a morphinan antagonist. Nature 2012;
485: 321–326.
61 Boivin S, Guilhaudis L, Milazzo I, Oulyadi H, Davoust D, Fournier A.
Characterization of urotensin-II receptor structural domains involved
in the recognition of U-II, URP, and urantide. Biochemistry 2006;
45: 5993–6002.
62 Wu B, Chien EY, Mol CD, Fenalti G, Liu W, Katritch V, Abagyan R,
Brooun A, Wells P, Bi FC, Hamel DJ, Kuhn P, Handel TM,
Cherezov V, Stevens RC. Structures of the CXCR4 chemokine GPCR
with small-molecule and cyclic peptide antagonists. Science 2010;
330: 1066–1071.
Supporting Information
46 Egloff P, Hillenbrand M, Klenk C, Batyuk A, Heine P, Balada S,
Schlinkmann KM, Scott DJ, Schuetz M, Plueckthun A. Structure of
signaling-competent neurotensin receptor 1 obtained by directed
evolutioninEscherichiacoli.Proc.Natl.Acad.Sci.U.S.A.2014;111:E655–E662.
Additional supporting information may be found in the online
version of this article at the publisher’s web site.
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015