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Orthosteric Binding of r-Da1a, a Natural Peptide of
Snake Venom Interacting Selectively with the a
1A
-
Adrenoceptor
Arhamatoulaye Maı
¨
ga
1
, Jon Merlin
2,3
, Elodie Marcon
1
,Ce
´
line Rouget
1
, Maud Larregola
1
,
Bernard Gilquin
4
, Carole Fruchart-Gaillard
1
, Evelyne Lajeunesse
1
, Charles Marchetti
5
, Alain Lorphelin
5
,
Laurent Bellanger
5
, Roger J Summers
2,3
, Dana S Hutchinson
2,3
, Bronwyn A Evans
2,3
, Denis Servent
1
,
Nicolas Gilles
1
*
1 Commissariat a
`
l’e
´
nergie atomique et aux e
´
nergies alternatives, iBiTec-S, Service d’Inge
´
nierie Mole
´
culaire des Prote
´
ines, Gif sur Yvette, France, 2 Department of
Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia, 3 Drug Discovery Biology, Monash Institute of Pharmaceutical
Sciences, Monash University, Parkville, Victoria, Australia, 4 Commissariat a
`
l’e
´
nergie atomique et aux e
´
nergies alternatives, iBiTec-S, Service de Bioe
´
nerge
´
tique, Biologie
Structurale et Me
´
canismes, Gif sur Yvette, France, 5 Commissariat a
`
l’e
´
nergie atomique et aux e
´
nergies alternatives, iBEB, Service de Biochimie et Toxicologie Nucle
´
aire,
Bagnols-sur-Ce
`
ze Cedex, France
Abstract
r-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the a
1A
-adrenoceptor. This toxin has
atypical pharmacological properties, including incomplete inhibition of
3
H-prazosin or
125
I-HEAT binding and
insurmountable antagonist action. We aimed to clarify its mode of action at the a
1A
-adrenoceptor. The affinity (pKi 9.26)
and selectivity of r-Da1a for the a
1A
-adrenoceptor were confirmed by comparing binding to human adrenoceptors
expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that r-Da1a, prazosin
and HEAT compete at the a
1A
-adrenoceptor. r-Da1a did not affect the dissociation kinetics of
3
H-prazosin or
125
I-HEAT, and
the IC
50
of r-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used,
while the residual binding by r-Da1a remained stable. The effect of r-Da1a on agonist-stimulated Ca
2+
release was
insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding
pocket of the a
1A
-adrenoceptor were evaluated for alterations in r-Da1a affinity. The D106
3.32
A and the S188
5.42
A/S192
5.46
A
receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86
2.64
A, F288
6.51
A and
F312
7.39
A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86
2.64
was identified as a key
interaction point for
125
I-HEAT, as the variant F86
2.64
A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic
acetylcholine receptor toxin MT7, r-Da1a interacts with the human a
1A
-adrenoceptor orthosteric pocket and shares
receptor interaction points with antagonist (F86
2.64
, F288
6.51
and F312
7.39
) and agonist (F288
6.51
and F312
7.39
) ligands. Its
selectivity for the a
1A
-adrenoceptor may result, at least partly, from its interaction with the residue F86
2.64
, which appears to
be important also for HEAT binding.
Citation: Maı
¨
ga A, Merlin J, Marcon E, Rouget C, Larregola M, et al. (2013) Orthosteric Binding of r-Da1a, a Natural Peptide of Snake Venom Interacting Selectively
with the a
1A
-Adrenoceptor. PLoS ONE 8(7): e68841. doi:10.1371/journal.pone.0068841
Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America
Received January 23, 2013; Accepted June 1, 2013; Published July 25, 2013
Copyright: ß 2013 Maı
¨
ga et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Nicolas.gilles@cea.fr
Introduction
Many toxins that interact with voltage- and ligand-gated ion
channels display both high affinity and selectivity. For the last 50
years, these properties have been used to identify, purify and
classify membrane targets and for structure/function studies. The
particular properties of these toxins are now also being exploited
pharmacologically, and some toxins are used as drugs and others
are currently undergoing preclinical trials [1–4].
Although voltage- and ligand-gated ion channels are the main
targets for neurotoxins, other targets, including G Protein-Coupled
Receptors (GPCRs), have also been identified. The animal toxins
active on GPCRs can be divided into two families [5]. Members of
the first family, the sarafotoxins, conopressin or contulakin-G
mimic the natural agonist of the targeted receptor: endothelin,
vasopressin and neurotensin, respectively. The second family
consists of highly reticulated toxins with folds that are unrelated to
any natural ligands. Nine have been isolated from mamba venoms
and are active against muscarinic acetylcholine receptors and
adrenoceptors (ARs), [6]. Two other toxins: r-TIA, from Conus
tulipa and b-cardiotoxin, from the snake Ophiophagus hannah, are
active against a
1
-ARs [7] and b-ARs [8], respectively. We
suspected that animal venoms are a potential source of novel
GPCR binding agents, and developed a screening strategy,
initially focused on the binding of green mamba venom to ARs.
This screening led to the isolation of two novel snake toxins from
Dendroaspis angusticeps: r-Da1a, previously called AdTx1, which is
highly selective for the a
1A
-AR [9], and r-Da1b, selective for a
2
-
ARs [10]. r-Da1a and r-Da1b are peptides of 65 and 66 residues,
PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e68841
respectively, reticulated by four disulfide bridges, and are members
of the three-finger-fold toxin family. The modes of action of these
peptide ligands on ARs are not clear. In equilibrium binding
experiments, neither r-Da1a nor r-Da1b fully inhibits radioligand
binding [9],[10]. In addition, in isolated prostatic muscle, r-Da1a
acts as an insurmountable antagonist [9], and cell-based assays
indicate that r-Da1b is a non-competitive antagonist at the human
a
2A
-AR [10].
Adrenergic and muscarinic toxins isolated from mamba snake
venoms belong to the same three-finger-fold family and display
substantial sequence identity (52–97%) [5]. The interactions
between MT1, MT7 and M1 muscarinic receptors have been
studied in detail [11–14]. Pharmacological studies indicate
competition between MT1 and
3
H-N- methylscopolamine
[11,14,15]. In contrast, MT7 significantly affects the dissociation
kinetics of
3
H-N- methylscopolamine and
3
H-acetylcholine [14,16]
and leaves residual binding in equilibrium binding experiments
[11] suggesting an allosteric mode of action. As a negative
allosteric modulator, MT7 reduces the efficacy and potency of
carbamylcholine at M1 muscarinic receptors expressed in CHO
cells [16] and interacts mainly with the extracellular loop 2 of this
receptor [13,17]. The smallest peptide ligand acting at ARs, r-
TIA, is a 19-residue toxin from Conus tulipa , and has been classified
as a non-competitive a
1B
-AR antagonist that accelerates
3
H-
prazosin dissociation kinetics and antagonizes a
1B
-AR activation
by an insurmountable mechanism [7]. A recent experimentally-
based model shows that r-TIA interacts primarily with extracel-
lular loop 3 (ec3) of the a
1B
-AR, consistent with its allosteric
properties [18]. Thus, both MT7 and r-TIA display a negative
allosteric mode of action by interacting with extracellular loops of
their receptor targets, namely ec2 of the M1 AChR, and ec3 of the
a
1B
-AR. r-TIA, however, shows only 10 to 25-fold selectivity for
the a
1B
-AR over the other a
1
-AR subtypes, and has been
described as a competitive antagonist at the a
1A
-AR although it
does not fully inhibit
125
I-HEAT binding [19].
These observations have led to hypotheses regarding the mode
of action of these peptide toxins at receptor targets. The aims of
our study were to use equilibrium and kinetic binding experiments
to establish the pharmacological behavior of r-Da1a at the a
1A
-
AR, to define the effect of r-Da1a on agonist-stimulated Ca
2+
release, and to use site-directed mutagenesis to analyze the a
1A
-AR
binding site for this peptide toxin.
Experimental Procedures
125
I-HEAT,
3
H-prazosin,
3
H-rauwolscine and
3
H-CGP-12177
were purchased from PerkinElmer (Courtaboeuf, France). Non
radioactive HEAT was obtained from Tocris (Ellisville, Missouri,
USA), and 5-(N-ethyl-N-isopropyl-amiloride (EPA), prazosin,
yohimbine, and propranonol were obtained from Sigma-Aldrich
(St Quentin-Fallavier, France).
Protein quantification
Total protein and membrane protein concentrations were
determined using the Bio-Rad protein assay, with bovine serum
albumin as standard.
Site-directed mutagenesis
a
1A
-AR cDNA inserted in the prK5 vector was kindly provided
by Michael Brownstein (Craig Venter Institute, Rockville, MD).
Point mutations were introduced into the a
1A
-AR gene by sense
and antisense primers (Sigma-Aldrich, St Quentin-Fallavier,
France) containing the desired changes, using the QuikChange
Site-Directed Mutagenesis kit. The incorporation of each mutation
was verified by DNA sequencing. The variants F308
7.35
A and
F312
7.39
A were generous gifts from Dr. Diane Perez (The
Cleveland Clinic Foundation, Cleveland, Ohio, USA).
Cell culture and membrane preparation
CHO cells stably expressing a
1
-ARs were kindly provided by
Dr. Herve´ Paris (INSERM U858, Toulouse, France) and were
grown in a 50:50 Dulbecco’s Modified Eagle’s Medium (DMEM)/
Ham’s F12 medium supplemented with 10% (v/v) foetal bovine
serum (FBS), glutamine (2 mM), penicillin (100 units/ml) and
streptomycin (100
mg/ml) at 37uC with 5% CO
2
. COS-7 cells
were grown at 37uC under 5% CO
2
in Dulbecco’s modified
Eagle’s medium containing 10% fetal calf serum, 1% penicillin
and 1% glutamine (Sigma-Aldrich, St Quentin-Fallavier, France).
At 80% confluence, the cells were transfected using a calcium
phosphate precipitation method for transient expression of the
genetic construct. After 48 h incubation at 37uC, cells were
harvested and the membranes were prepared as follow. Cells were
washed with ice-cold phosphate buffer and centrifuged at 1700 g
for 10 min (4uC). The pellet was suspended in ice-cold buffer
(1 mM EDTA, 25 mM sodium phosphate, and 5 mM MgCl
2
,
pH 7.4) and homogenized using an Potter-Elvehjem homogenizer
(Fisher Scientific Labosi, Elancourt, France). The homogenate was
centrifuged at 1700 g for 15 min (4uC). The sediment was
resuspended in buffer, homogenized, and centrifuged at 1700 g
for 15 min (4u C). The combined supernatants were centrifuged at
35,000 g for 30 min (4uC), and the pellet was suspended in the
same buffer (0.1 ml/dish). The CHO cells used for Ca
2+
release
experiments also stably express the human a
1A
-AR (B
max
531694 fmol/mg protein, pK
D
for
125
I-HEAT 9.260.09 [20].
Cells were grown in a 50:50 Dulbecco’s Modified Eagle’s Medium
(DMEM)/Ham’s F12 medium supplemented with 10% (v/v)
foetal bovine serum (FBS), glutamine (2 mM), penicillin (100
units/ml) and streptomycin (100
mg/ml) at 37uC with 5% CO
2
.
Media was changed every 2–3 days and cells were passaged when
confluent with 0.05% trypsin and 0.02% EDTA.
Binding assays
We used
3
H-prazosin and
125
I-HEAT (all incubations were
done in the dark) as selective ligands for a
1
-ARs,
3
H-rauwolscine
for a
2
-ARs and
3
H-CCGP-12177 for b-ARs. Non-specific binding
to a
1
, a
2
and b-ARs was measured in presence of prazosin
(10
mM), yohimbine (10 mM) and propanolol (10 mM), respective-
ly. Binding experiments were performed in a 100
mL reaction mix
at room temperature in buffer composed of 50 mM Tris-HCl,
pH 7.4, 10 mM MgCl
2
, 1 g/L BSA. Reactions were stopped by
filtration through 96 GF/C filter plates pre-incubated with 0.5%
polyethylenimine. An aliquot of 25
mL of Microscint 0 was added
onto each dry filter and the radioactivity was quantified on a
TopCount beta counter with a 33% yield (PerkinElmer,
Courtaboeuf, France). Saturation binding assays were performed
using a fixed amount of receptors and a series of concentrations of
125
I-HEAT with an incubation time of 1 h. Competition binding
assays were performed by mixing the radioligand (2 nM of
3
H-
prazosin or
3
H-rauwolscine, 0.2–1.3 nM of
125
I-HEAT, 6 nM of
3
H-CGP-12177) with a range of competitor concentrations before
adding membranes (a
1A
-AR: 1 mg for
3
H-prazosin and 0.1 mg for
125
I-HEAT, a
1B
:3mg, a
1D
:29mg, a
2A
: 140 mg; a
2B
: 100 mg, a
2C
:
3
mg, b
1
:3mg, b
2
: 1.5 mg, or a
1A
-mutants: 0.1–1 mg), for 16 h of
incubation. Dissociation kinetics experiments were performed by
pre-equilibrating
125
I-HEAT (400 pM) or
3
H-prazosin (2 nM) for
3 hours with a
1A
-AR COS-7 cell membranes (0.2 or 1 mg,
respectively). Radiotracer dissociation was then measured follow-
ing addition of HEAT (5
mM) or prazosin (10 mM) alone or with
Binding Mode of an Adrenoceptor-Selective Toxin
PLOS ONE | www.plosone.org 2 July 2013 | Volume 8 | Issue 7 | e68841
r-Da1a (2.5 mM), 5-(N-ethyl-N-isopropyl)-amiloride (EPA,
150
mM) or adrenaline (2 mM).
Measurement of intracellular Ca
2+
concentration
CHO-K1 cells expressing the a
1A
-AR were seeded at 2610
4
cells per well in 96-well plates overnight. The following morning,
the media was removed and cells washed three times in a modified
Hanks’ buffered saline solution (HBSS; composition in mM: NaCl
150, KCl 2.6, MgCl
2
.2H
2
O 1.18, D-glucose 10, Hepes 10,
CaCl
2
.2H
2
O 2.2, probenecid 2, pH 7.4) containing BSA 0.5%
(w/v). In light-diminished conditions cells were treated with fluoro-
4 (0.1% v/v in modified HBSS, 1 h, 37uC). Excess fluoro-4 not
taken up by the cells was removed by washing twice in modified
HBSS and then cells incubated for a further 30 min in the absence
or presence of differing concentrations of r-Da1a before the assay
plate was transferred to a FlexStation (Molecular Devices, Palo
Alto CA, USA). Real-time fluorescence measurements were
recorded every 1.7 seconds over 200 seconds, with agonist
(noradrenaline, phenylephrine, A61603 or oxymetazoline) addi-
tions occurring after 17 seconds, using an excitation wavelength of
485 nm and reading emission wavelength of 520 nm. All
experiments were performed in duplicate. Agonist responses
represent the difference between basal fluorescence and peak
[Ca
2+
]i measurements expressed as a percentage of the response to
A23187 (1
mM) in each experiment.
Data analysis
Binding data were analyzed by nonlinear regression using the
KaleidaGraph 4.0 software (Synergy software, Reading, PA). pK
D
values and Bmax (number of binding sites) were determined by
applying a nonlinear regression to data obtained with saturation
binding assays. The nonlinear regression used was
BS = (Bmax*A)/K
D
+A, where BS is the specific binding, Bmax
the number of binding sites, A the concentration of radioligand,
and K
D
the dissociation constant of the radioligand. Data resulting
from competition binding assays were analyzed using the Hill
equation for IC
50
and curve slope estimations. The binding affinity
(pK
i
)ofr-Da1a was determined from the IC
50
value of inhibition
curves using the Cheng and Prussof equation [21]. The linear
curves were analyzed with IC
50
=K
i
+(L/K
D
)*K
i
. Dissociation
kinetics were analyzed using a simple equation of exponential
decay BS * exp (-K
off
*t), where BS is the specific binding at time
zero and K
off
is the dissociation rate constant. Results are
expressed as mean 6 s.e. mean from n independent experiments.
One-way Anova test was used to compare values. A p,0.05 was
accepted for statistical significance.
Values for intracellular Ca
2+
release are expressed as mean 6
s.e. mean from n independent experiments. Data were analysed
using non-linear curve fitting (Graph Pad PRISM v5.02) to obtain
pEC
15
values for the [Ca
2+
]i assays. Antagonists such as r-Da1a
that have slow dissociation kinetics are prone to display hemi-
equilibrium artifacts in functional transient responses such as
measurement of intracellular Ca
2+
levels. As such, when compet-
ing with an agonist, the maximal response achieved by the agonist
reduces in the presence of higher antagonist concentrations due to
the inaccessibility of a large pool of the receptors in the time taken
for the transient response to occur [22]. This affects the ability of a
Schild analysis to estimate the pK
B
of r-Da1a. In order to account
for this, the pK
B
value for r-Da1a was calculated by the modified
Lew-Angus method [23] using pEC
15
values, based on the extent
of reduction in agonist maximal responses in the presence of r-
Da1a. The pEC
15
values were plotted against the concentration of
antagonist and non-linear regression applied [23,24] to estimate
pK
B
values for r-Da1a against each of the four different agonists.
Homology modeling
A model of the a
1A
-AR was generated with MODELLER [25].
The receptor with the most similar sequence to the a
1A
-AR is the
b
2
-AR subtype, with an overall amino acid sequence identity of
21% [26,27], identity within 7TM domain from helix 1 to helix 8
(excluding intracellular ic3 loop) 38%, sequence similarity 61%
[BLASTP]. Nine b
2
-AR structures are available (2RH1, 3D4S,
3KJ6, 3NY8, 3NY9, 3NYA, 3PDS, 3P0G, 3SN6) and are very
similar (Ca RMSDs,1.5 A
˚
for 253 residues). We used the X-ray
structure with the highest resolution (2RH1) as a template [28].
Results
r-Da1a, (previously AdTx1) [9], was renamed according to a
rational nomenclature [29]. A recombinant expression system
producing the toxin with an extra glycine residue at its N-terminus
was developed (Figure S1 in File S1). Recombinant r-Da1a
displays the same affinity as the chemically synthesized toxin,
indicating that the N-terminal glycine has no consequences for
function. The pharmacological experiments reported in this study
were performed with the recombinant form of the toxin.
Selectivity of r-Da1a
r-Da1a affinity was recently determined in tissue preparations,
and using human and rat a
1
-ARs expressed in yeast [9]. To
complete the r-Da1a selectivity profile, we expressed human ARs
in eukaryotic cells and performed competition binding with
additional receptor subtypes. The pKi values derived from these
experiments [21] were: 9.1960.09 for a
1A
-ARs, 7.2860.09 for
a
1B
-ARs, 6.8560.08 nM for a
2C
-ARs, and 5.9560.08 for a
1D
-
ARs. No significant effect was observed with 10 mMofr-Da1a at
a
2A
, a
2B
, b
1
,orb
2
-ARs. For a
1A
- and a
1B
-ARs, even the highest
concentrations of r-Da1a did not completely inhibit
3
H-prazosin
binding, which remained at 1863% and 1761%, respectively
(Fig. 1).
Figure 1. Pharmacological profile of r-Da1a binding to various
human AR subtypes expressed in eukaryotic cells. Binding
inhibition curves for
3
H-prazosin (2 nM),
3
H-rauwolscine (2 nM) and
3
H-
CGP-12177 (6 nM) on ha
1A
-(1mg, #), ha
1B
-(3mg,
N
), ha
1D
- (29 mg, %),
ha
2A
- (140 mg, e), ha
2B
- (100 mg, D), ha
2C
-(3mg, x), b
1
-(3mg,.) and b
2
-
AR (1.5
mg, &) with recombinant r-Da1a. n = 4.
doi:10.1371/journal.pone.0068841.g001
Binding Mode of an Adrenoceptor-Selective Toxin
PLOS ONE | www.plosone.org 3 July 2013 | Volume 8 | Issue 7 | e68841
r-Da1a, prazosin and HEAT compete at a
1A
-ARs
3
H-prazosin binding was fully inhibited by HEAT (pKi
9.6660.08 nM, Hill slope 0.85, Fig. 2) and
125
I-HEAT binding
was fully displaced by prazosin (pKi 9.1860.07 nM, Hill slope
0.95). However, as observed with
3
H-prazosin, r-Da1a interacts
very efficiently with the a
1A
-AR (pKi 9.2660.07 nM, Hill slope
0.92, Fig. 2), but does not inhibit more than 80% of
125
I-HEAT
binding. This residual binding is stable with time, as we detected
no variation with incubation times from 2 to 24 hours (data not
shown).
Dissociation kinetic experiments are classically used to identify
negative allosteric modulators [30,31]. The influence of r-Da1a on
the dissociation kinetics of
3
H-prazosin and
125
I-HEAT was
studied in comparison with adrenaline and the negative allosteric
modulator EPA (Fig. 3).
3
H-prazosin dissociation from a
1A
-ARs
was mono-exponential and the dissociation rate was
0.0560.01 min
21
. Consistent with previous studies [32], this
value was increased 2.6 times in the presence of 150
mM EPA
(K
off+ EPA
= 0.15 min
21
). In contrast, neither 2 mM adrenaline
nor 2.5
mM r-Da1a affected the
3
H-prazosin dissociation rate
(K
off+adrenaline
= 0.054 min
21
;K
off+r-Da1a
= 0.059 min
21
, Fig. 3,
n = 2) in the presence of excess prazosin.
125
I-HEAT dissociation
rates were measured in the absence (K
off
= 0.062 min
21
) and in
the presence of r-Da1a (K
off HEAT+r-Da1a
= 0.058 min
21
), prazosin
(K
off HEAT+prazosin
= 0.06 min
21
), and EPA (K
off HEAT+EPA
=
0.37 min
21
, Fig. 3, n = 2): neither prazosin nor r-Da1a affected
the HEAT dissociation rate; whereas EPA increased the dissociation
rate by six-fold.
The r-Da1a IC
50
values were determined using various
concentrations of radiotracers in competition binding experiments
(Fig. 4). Eleven concentrations of
3
H-prazosin (0.2, 0.5, 1.0, 1.86,
3.55, 4.53, 8.0, 9.14, 10, 13 and 16 nM) were dose-dependently
inhibited by r-Da1a (IC
50
of 2.4, 2.9, 2.6, 3.55, 10.2, 5.54, 18, 14,
20, 25 and 31 nM) with Hill slopes between 0.8 and 1.1. Residual
binding in the presence of
3
H-prazosin fluctuated between 15 to
25% of the total binding, but did not show any trend to
concentration-dependence. The curve IC
50r-Da1a
as a function
of
3
H-prazosin concentration (L) fitted the linear regression
IC
50r-Da1a
= 1.067+1.82*L, incompatible with a negative allosteric
modulation (Fig. 4A). Using the equation IC
50r-Da1a
=Ki
r-Da1a
+
(Ki
r-Da1a
*L
prazosin
/Kd
prazosin
) [21], this experiment gave a Ki
r-Da1a
of 1.067 nM (pKi 8.97) and a Kd
prazosin
of 0.586 nM (pKd 9.23).
Figure 2. Inhibition of
3
H-prazosin (2 nM, 1 mg, open symbols)
by HEAT (%), and r-Da1a (circle), and inhibition of
125
I-HEAT
(0.2 nM, 0.2
mg, full symbols) binding by prazosin (¤) and r-
Da1a (circle) to a
1A
-AR. n=3.
doi:10.1371/journal.pone.0068841.g002
Figure 3. Influence of various ligands on
3
H-prazosin and
125
I-
HEAT dissociation. Panel A: Dissociation of
3
H-prazosin (2 nM)
binding to a
1A
-AR (1 mg) in the presence of prazosin (10 mM, black),
prazosin plus r-Da1a (2.5
mM, blue), prazosin plus adrenaline (2 mM,
red) and prazosin plus EPA (150
mM, green). Panel B : dissociation of
125
I-HEAT (0.4 nM) binding to a
1A
-AR (0.2 mg) in the presence of HEAT
(5
mM, black), HEAT plus r-Da1a (2.5 mM, blue), HEAT plus prazosin
(10
mM, red) and HEAT plus EPA (150 mM, green). n = 2.
doi:10.1371/journal.pone.0068841.g003
Binding Mode of an Adrenoceptor-Selective Toxin
PLOS ONE | www.plosone.org 4 July 2013 | Volume 8 | Issue 7 | e68841
An analogous experiment was performed using
125
I-HEAT. Ten
concentrations (0.1, 0.13, 0.2, 0.3, 0.38, 0.4, 0.5, 0.7, 0.9 and
1.25 nM) were inhibited by r-Da1a (IC
50
of 4.0, 2.75, 5.3, 3.23,
6.84, 8.0, 8.18, 11.5, 12.7 and 23.4 nM) with Hill slopes between
0.9 and 1.4. Residual binding fluctuated between 18 to 28% of the
total binding (Fig. 4B). IC
50r-Da1a
as a function of
125
I-HEAT
concentrations fitted the equation IC
50r-Da1a
= 0.706+16.22*L
which gave a Ki
r-Da1a
of 0.706 nM (pKi 9.15) and a Kd
HEAT
of
0.0435 nM (pKd 10.36).
Insurmountable antagonism of intracellular Ca
2+
release
by r-Da1a
We next tested the effect of r-Da1a on responses to
noradrenaline and phenylephrine (phenethylamine agonists) and
A61603 and oxymetazoline (imidazoline agonists). In CHO-K1
cells expressing the a
1A
-AR, all agonists stimulated Ca
2+
release
(Figure 5), with pEC
50
and E
max
values consistent with previous
work [20] – noradrenaline pEC
50
8.6360.08, E
max
(as a
percentage of peak A23187 response) 77.861.5, phenylephrine
pEC
50
7.6460.16, E
max
64.862.4, A61603 pEC
50
10.1760.07,
Figure 4. Inhibition of the binding of a series of concentrations of
3
H-prazosin and
125
I-HEAT to a
1A
-AR by r-Da1a. Panel A
3
H-prazosin
binding (from 0.2 to 16 nM) inhibited by r-Da1a. Panel B
125
I-HEAT binding (from 0.1 to 1.25 nM) inhibited by r-Da1a. Panel C and D: Fitting, by the
Cheng and Prusoff equation IC
50
=Ki+Ki(L/Kd), of IC
50
values as a function of the radiotracer concentrations.
doi:10.1371/journal.pone.0068841.g004
Binding Mode of an Adrenoceptor-Selective Toxin
PLOS ONE | www.plosone.org 5 July 2013 | Volume 8 | Issue 7 | e68841
E
max
75.561.3, and oxymetazoline pEC
50
9.0960.08, E
max
60.861.3. Increasing concentrations of r-Da1a reduced the
maximal response to each of the four agonists while shifting the
curves to the right. The pK
B
values for r-Da1a were calculated by
a modified Lew-Angus method [23] and were similar irrespective
of the agonist employed (7.7160.05 vs noradrenaline; 7.6060.04
vs phenylephrine; 7.6660.10 vs A61603; 7.6760.05 vs oxyme-
tazoline).
Molecular characterization of the r-Da1a/a
1A
-AR
interaction
We investigated the involvement of residues within the
orthosteric pocket of a
1
-ARs that are known to interact with
agonists and/or antagonists: F86
2.64
[33], D106
3.32
(D125 in a
1B
-
AR) [34–36], F187
5.41
[37], S188
5.42
and S192
5.46
[38], F288
6.51
(F310 on a
1B
-AR) [18,39], M292
6.55
[40] and F308
7.35
and
F312
7.39
[18,41] (superscripts refer to the Ballesteros-Weinstein
numbering system for residues in 7TM helices). In addition, we
tested the positions F193
5.47
and F281
6.44
, predicted to play a role
in the stabilization of the active state of a
1A
-AR [42] (Table 1). In
saturation binding experiments,
125
I-HEAT affinity values for
D106
3.32
A, F193
5.47
A, F281
6.44
A, F288
6.51
A, M292
6.55
A and
F308
7.35
A were not significantly different from the wild type
receptor. One mutated receptor, F187
5.41
A, had significantly
higher affinity for
125
I-HEAT with a pK
D
of 10.7060.005
compared to wild type pK
D
10.0560.09 (Table 1, p,0.05). Only
the F86
2.64
A mutant showed a substantial 23-fold loss of
125
I-
HEAT affinity (pK
D
8.6860.09, p,0.05, Fig. 6, Table 1). The
transiently transfected mutant receptors showed marked variability
in expression level, with B
max
values ranging from 0.63 up to
29 pmol/mg protein compared to 11.3 pmol/mg protein for the
wild type a
1A
-AR (Table 1), however there was no correlation
between receptor abundance and the observed binding affinity of
125
I-HEAT. For example, D106
3.32
A (0.63 pmol/mg protein) and
F308
7.35
A (29 pmol/mg protein) both displayed a similar pKd to
each other and to the wild type receptor.
Curves for competition of HEAT and r-Da1a with binding of
125
I-HEAT are shown at wild type, D106
3.32
A and F86
2.64
A
receptors (Figure 7). As seen in the saturation binding experiment,
HEAT had similar affinity for the D106
3.32
A variant (pKi
9.7460.12) and the wild type receptor (pKi 9.5760.08) but was
strongly affected by the F86
2.64
A mutation (pKi 8.2160.09). r-
Da1a affinity at the D106
3.32
A variant was reduced by 6-fold (pKi
8.4860.11) compared to the wild type receptor while affinity at
F86
2.64
A was reduced by 36-fold (pKi 7.7060.06). The mutation
F86
2.64
A affects both HEAT and r-Da1a affinities, suggesting that
the structural organization of the receptor could have been
perturbed by this modification. We used the radioligand
3
H-
prazosin to examine this point and found that prazosin affinity for
the F86
2.64
A mutant (pKd 9.2160.07) was very close to the one
measured for wild type receptor (9.2660.05; data not shown).
While this mutation may alter interactions between F86
2.64
and
other aromatic residues within the orthosteric pocket [43], the
Figure 5. Concentration-response curves for stimulation of Ca
2+
release by the a
1A
-AR. Agonist responses represent the difference
between basal fluorescence and the peak [Ca
2+
]i (reached within 20 sec of agonist addition), expressed as a percentage of the response to the Ca
2+
ionophore A23187 (1 mM). Concentration-dependent Ca
2+
release was stimulated by noradrenaline (panel A), phenylephrine (panel B), A61603 (panel
C) or oxymetazoline (panel D). Concentration response curves were performed in the presence or absence of differing concentrations of r-Da1a (
N
control, & 1 nM, m 3 nM, . 10 nM, ¤ 30 nM, e 100 nM, # 300 nM). Values are means 6 SEM of 3–4 independent experiments.
doi:10.1371/journal.pone.0068841.g005
Binding Mode of an Adrenoceptor-Selective Toxin
PLOS ONE | www.plosone.org 6 July 2013 | Volume 8 | Issue 7 | e68841
retention of prazosin affinity indicates that there is no global effect
on the native structural conformation of the receptor. As seen for
the wild type a
1A
-AR, r-Da1a inhibited only 75–80% of
125
I-
HEAT binding in experiments with these mutant receptors.
r-Da1a affinities were tested on eight additional receptor
variants (Fig. 8). At the F187
5.41
A, M292
6.55
A and F308
7.35
A
variants, r-Da1a inhibited
125
I-HEAT binding with affinities
similar to the wild type (Table 1). However the F288
6.51
A and the
F312
7.39
A variants decreased r-Da1a affinity by 18 and 93 times,
with pKi of 8.0060.08 and 7.2860.10, respectively. Again, r-
Da1a left residual binding between 20 and 30%, except for the
F187
5.41
A variant (963%) receptor (Fig. 8).
Discussion
r-Da1a is the first natural peptide shown to be selective for the
a
1A
-AR. Due to its high selectivity and potent relaxing effect on
isolated prostate smooth muscle [5,9,44], the peptide is in the
process of therapeutic development. In recombinant expression
systems, r-Da1a can be produced with a final yield of 5 mg per
liter of culture. The recombinant toxin interacts with the a
1A
-AR
in a similar manner to the chemically synthesized one.
The selectivity profile of r-Da1a for human ARs was
established, and confirmed a sub-nanomolar affinity for the a
1A
-
AR subtype: the order of selectivity is a
1A
.a
1B
.a
2C
.a
1-
D
..a
2A
= a
2B
= b
1
= b
2
. The affinities of r -Da1a for a
1
-ARs
expressed in yeast or in mammalian cells were slightly different:
0.35 and 0.55 nM for a
1A
-AR, 420 nM and 1110 nM for a
1D
-AR,
and 317 and 53 nM for a
1B
-AR respectively. These differences in
toxin affinity may be related to differences in associated lipids or
proteins in the membranes of these cells, as described for the m-
opioid [45] or the dopamine D
2S
receptors [46].
We have characterized the interaction between r-Da1a and the
a
1A
-AR by a series of binding and functional experiments. Our
findings from Ca
2+
release assays, competition binding and
radioligand dissociation curves in the presence of r-Da1a generally
indicate competition between the toxin and small molecule
ligands. On the other hand, r-Da1a was unable to completely
inhibit orthosteric radioligand binding to a
1A
-ARs regardless of
the time of incubation (2 to 24 h), the radioligand (
3
H-prazosin or
125
I-HEAT), or the expression system (yeast, CHO, or COS-7
cells), a finding more consistent with non-competitive interaction.
To examine functional antagonism by r-Da1a, we measured
blockade of intracellular Ca
2+
release following 30 min pre-
incubation of CHO-a
1A
-AR cells with the toxin (Fig. 5). The
observed insurmountable antagonism indicates that at higher
concentrations of r-Da1a, a large proportion of receptors are
inaccessible to agonist during the time taken for the transient Ca
2+
response [22]. This effect is in part due to the slow dissociation
kinetics of r-Da1a [9], which prevents the system from reaching
equilibrium under the assay conditions [22]. The reduction in
E
max
is governed by the efficacy of each agonist – for example
oxymetazoline is a high affinity, low efficacy agonist that displays a
greater loss of maximal response in the presence of r-Da1a than
high efficacy agonists such as noradrenaline and A61603.
Essentially there is lower receptor reserve for responses to
oxymetazoline than to noradrenaline or A61603. Despite these
differences in reduction of E
max
, the pK
B
values for r-Da1a
blockade of Ca
2+
release remained the same irrespective of the
agonist used (between 7.6 and 7.71). These data conform to ‘‘non-
permissive’’ antagonism, where receptor occupancy by the toxin
prevents simultaneous orthosteric agonist interaction [47]. In the
converse situation where a toxin (for example MT7) binds to a
receptor at a site distinct from the orthosteric pocket, receptors are
able to bind simultaneously both the toxin and an agonist –
illustrating ‘‘permissive’’ antagonism characteristic of allosteric
modulators [48]. As different agonists adopt distinct poses in the
orthosteric binding site, they have the capacity to differentially
affect the affinity of an allosteric modulator for the receptor, thus
pK
B
values of the modulator are altered depending on the agonist
used [48,49]. Our finding that the pK
B
of r-Da1a is the same for
four agonists belonging to two distinct structural classes, and
known to display signaling bias at the a
1A
-AR [20], corroborates
our other data showing that r-Da1a has no effect on the
dissociation rate of either
3
H-prazosin or
125
I-HEAT, and that
r-Da1a affinity for the a
1A
-AR is reduced by mutation of residues
within the orthosteric pocket.
Figure 6. Saturation experiments with
125
I-HEAT on receptor
variants. Total (black), specific (green) and non specific (red) binding of
125
I-HEAT to: panel A wild-type receptor (0.2 mg, open symbols) and
F86
2.64
A variant (0.8 mg, full symbols). Panel B: D106
3.32
A(1mg, open
symbol) and F312
7.39
A variant (0.8 mg, full symbols), n = 3.
doi:10.1371/journal.pone.0068841.g006
Binding Mode of an Adrenoceptor-Selective Toxin
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Allosteric modulators are generally characterized by effects on
the dissociation rate of orthosteric radioligands in kinetic binding
experiments. For example, MT7 significantly affects the dissoci-
ation kinetics of
3
H-N-methylscopolamine and
3
H-acetylcholine in
membranes expressing the M1 AChR [14,16], and the negative
allosteric modulator EPA (5-(N-ethyl-N-isopropyl-amiloride) sub-
stantially increases the dissociation rate of both
3
H-prazosin and
125
I-HEAT from the a
1A
-AR (Fig. 3 in this study, [32]). In
contrast, r-Da1a has no effect on the dissociation rate of
3
H-
prazosin or
125
I-HEAT (Fig. 3). Reciprocally, prazosin has no
effect on the
125
I-rDa1a dissociation rate [9], indicating compet-
itive behavior. In equilibrium binding experiments, there was a
linear relationship between the IC
50
of r-Da1a and the
concentration of
3
H-prazosin or
125
I-HEAT (Fig. 4, panel C and
D), again consistent with competitive behavior [50].
The observed competition between r-Da1a and radioligands at
the a
1A
-AR suggested that the toxin directly interacts with the
orthosteric binding pocket. To provide further evidence for this
proposal, ten positions belonging to the orthosteric pocket of the
a
1A
-AR were tested for effects on r-Da1a affinity. Previous studies
on the a
1A
-AR have shown that residues F187
5.41
[37] and
M292
6.55
[40] are important for agonist and/or antagonist
binding, and the two residues F193
5.47
and F281
6.44
[42]
Table 1. Effect of human a
1A
-AR mutations on receptor expression and affinity for HEAT and r-Da1a.
Variant Position
125
I- HEAT HEAT r-Da1a
Bmax pmol/mg pK
d
Ratio pKi ratio pKi Ratio
WT 11.362.3 10.0560.14 1 9.5760.08 1 9.2660.07 1
F86A 2.64 22.662.4 8.6860.09 23* 8.2160.09 23* 7.7060.06 36*
D106A 3.32 0.6360.05 9.8260.11 1.7 9.7460.12 0.67 8.4860.11 6.0*
F187A 5.41 20.563.5 10.7060.01 0.22* 9.1260.07 1.4
SS-AA 5.42–5.46 13.363.3 10.1560.01 0.78 8.3860.09 7.6*
F193A 5.47 11.562.2 9,6060.09 2.8 9.6460.11 0.42
F281A 6.44 18.264.8 9.4660.09 3.9 9.6660.09 0.40
F288A 6.51 14.362.5 9.9260.12 1.3 8.0060.08 18*
M292A 6.55 15.863.5 9.6960.09 2.2 9.4160.11 0.71
F308A 7.35 2964.2 9.6660.11 2.4 9.1560.07 1.3
F312A 7.39 3.260.12 10.4060.10 0.44 7.2860.10 93*
*for p,0.05. Position refers to the Ballesteros-Weinstein numbering scheme for residues within TM domains of G protein-coupled receptors. n = 3–6.
doi:10.1371/journal.pone.0068841.t001
Figure 7. Receptor affinities for r-Da1a (dash lines) and HEAT
(solid lines) on mutated a
1A
-ARs. Binding inhibition curves for
125
I-
HEAT binding to WT (200 pM, 0.2
mg, #), D106
3.32
A (200 pM, 1 mg, %)
and F86
2.64
A (1.3 nM, 0.8 mg,
N
) receptor variants. n = 3–4.
doi:10.1371/journal.pone.0068841.g007
Figure 8. Receptor affinities for r-Da1a on mutated a
1A
-ARs.
Binding inhibition curves for
125
I-HEAT (200 pM) binding to WT (0.2 mg,
black), F187
5.41
A (0.15 mg, light blue), the double S188
5.42
,S192
5.46
-AA
(0.3
mg, dark blue), F193
5.47
A (0.25 mg, green), F281
6.44
A (0.15 mg,
orange), F288
6.51
A (0.2 mg, red), M292
6.55
A (0.2 mg, purple), F308
7.35
A
(0.1
mg, brown), F312
7.39
A (0.8 mg, grey), n = 3–4.
doi:10.1371/journal.pone.0068841.g008
Binding Mode of an Adrenoceptor-Selective Toxin
PLOS ONE | www.plosone.org 8 July 2013 | Volume 8 | Issue 7 | e68841
participate in stabilizing the active conformation of the receptor.
Aside from an increase of 4.5 fold in HEAT affinity at the
F187
5.41
A variant, none of these mutations affected either r-Da1a
or HEAT affinity (Table 1).
The negative charge of D106
3.32
is expected to interact with the
positive charge of biogenic amines [51], and no binding of
3
H-
prazosin to the a
1A
-AR variants D106
3.32
A or D106
3.32
A/N167F
is observed [36]. The homologous D125
3.32
A variant of the a
1B
-
AR has been expressed but showed no change in affinity for
HEAT [52], although another study has shown a total loss of
affinity for HEAT [34]. In our study, HEAT affinity was not
affected by alanine substitution of residue D106
3.32
, whereas toxin
affinity was reduced six-fold. The TM5 residues S188
5.42
/S192
5.46
are one helical turn apart, and have been implicated in agonist
binding and receptor activation. While either single mutation,
S188
5.42
A or S192
5.46
A does not alter agonist binding, the double
mutation reduces agonist affinity [38]. In our hands, the double
mutation moderately reduced r-Da1a affinity by 7.6-fold.
We found three key mutations that have major importance for
r-Da1a binding: F86
2.64
A, F288
6.51
A and F312
7.39
A. Residue
F312
7.39
in the a
1A
-AR has been described to interacts with
prazosin and imidazoline-type agonists [41]. Phenylalanine F310
at position 6.51 in the a
1B
-AR (F288 in the a
1A
-AR) is a major
determinant for the interaction with the aromatic ring of
catecholamines and with a
1
-AR antagonists like prazosin and
phentolamine [18,39]. Thus, r-Da1a shares two major interaction
points with prazosin, F288
6.51
and F312
7.39
, and one with
phenethylamine-type (F288
6.51
) and imidazoline-type (F312
7.39
)
agonists. In addition, F86
2.64
is the only residue unique to the a
1A
-
AR subtype that is important for toxin affinity. In a
1B
-, a
2C
- and
a
1D
-ARs, this position is occupied by a leucine, an asparagine and
a methionine, respectively. F86
2.64
was previously identified as a
determinant for interaction of the a
1A
-AR with various antagonists
[33]. While a F86
2.64
M receptor mutant did not show any changes
in HEAT affinity [33], the F86
2.64
A one strongly decreased it
while having no effect on prazosin affinity. This residue most likely
contributes to the selectivity of r-Da1a for the a
1A
-AR subtype.
We constructed a homology model of the a
1A
-AR based on the
b
2
-AR structure [28]. Green, orange and red denote residues with
no, moderate or large influence on r-Da1a affinity (Fig. 9, panel A
and B). Residue D106
3.32
and the S188
5.42
/S192
5.46
positions are
about 16 A
˚
from the surface of the receptor on one side of the
orthosteric site, whereas positions F86
2.64
, F288
6.51
and F312
7.39
that all interact strongly with r-Da1a are located on the opposite
side and distributed from the surface down to a depth of 12 A
˚
.As
seen for toxin binding, mutation of F86
2.64
had a substantial effect
on HEAT affinity, whereas mutation of F288
6.51
had no effect,
and mutation of F312
7.39
to alanine caused a slight increase in the
affinity of HEAT. Although we have yet to define additional
residues that contribute to HEAT binding, these findings support
the idea that r-Da1a and the radioligands have overlapping but
distinct binding modes.
A three-finger fold toxin can be represented by a 35 A
˚
isosceles
triangle of around 10 A
˚
thickness (Fig. 9C). It is therefore much
larger than classical small orthosteric ligands but nevertheless, r-
Da1a is able to interact with positions within the orthosteric cavity
Figure 9. Homology modelling of the r-Da1a binding site in the
a
1A
-AR and the MT7 toxin. Views from the side of the TM bundle
(Panel A), and from the top of the extracellular space (Panel B). F187
5.41
,
F193
5.47
, F281
6.44
, M292
6.55
, F308
7.35
in green. D106
3.32
and the double
S188
5.42
/S192
5.46
in orange. F86
2.64
, F288
6.51
and F312
7.39
in red. Panel C
:3D structure of the three-finger fold MT7 toxin (2vlw) with the four
conserved disulfide bridges in red.
doi:10.1371/journal.pone.0068841.g009
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PLOS ONE | www.plosone.org 9 July 2013 | Volume 8 | Issue 7 | e68841
of the a
1A
-AR. This is not the case for MT7, as the
experimentally-based model of the MT7-M1 muscarinic receptor
complex indicates an extracellular location of the toxin involving
mainly the extracellular loop e2, in agreement with its allosteric
properties [13]. The structural organization of the external part of
GPCRs plays an important role in the access to the orthosteric site
by agonists. Some receptors, like rhodopsin [53] or the S1P
1
receptor [54] have their ligand-binding cavity substantially
enclosed, compared to the chemokine CXCR4 receptor, for
example, in which the extracellular loop conformation renders the
binding cavity particularly open, facilitating the binding of large
peptides [55]. The top view of the a
1A
-AR model (Fig. 9B) shows a
relatively open receptor with external loops on the sides of the
receptor, very similar to that proposed for the a
1B
-AR [18]. By
comparison, M1 receptor modeling and mutational analysis
indicate that extracellular loop 2 is important in binding of both
orthosteric and allosteric ligands. The loop shows conformational
flexibility but adopts closed conformations that affect access even
of small molecule ligands [56]. Residues E170, R171, L174 and
Y179 located in the e2 loop of the human M1 receptor collectively
interact with MT7, with additional contributions from W91 in the
e1 loop and W400 at the top of TM7 [13]. The more open
conformation of the a
1A
-AR is certainly consistent with the
capacity of r-Da1a to interact with residues inside the orthosteric
pocket, however the large size and sub-nanomolar affinity of the
toxin also suggest additional points of interaction with the
receptor. A very recent publication describes how r-TIA, a
conotoxin of 19 residues which acts as a negative allosteric
modulator, interacts with the a
1B
-AR [18]. This small reticulated
peptide binds primarily with the extracellular loop e3 of the a
1B
-
AR and with the upper part of TM6 and TM7. r-TIA affinity is
increased by the mutation F310
6.51
A in TM6, whereas our
homologous mutation in the a
1A
-AR (F288
6.51
A) decreases r-
Da1a affinity 18-fold. In TM7, r-TIA is sensitive to mutation at
position F330
7.35
of the a
1B
-AR (corresponding to F308
7.35
in a
1A
-
AR, not implicated in r-Da1a affinity) but not at position F334
7.39
(corresponding to F312
7.39
in a
1A
-AR). Mutation of the a
1A
-AR at
residue F312
7.39
, which is one helical turn further from the
extracellular face of the a
1A
-AR than F308
7.35
, produces a 93-fold
reduction in r-Da1a affinity, highlighting the difference in binding
mode of r-Da1a and the a
1A
-AR compared to r-TIA and the a
1B
-
AR. Hence of the three animal toxins for which the mode of action
has been described, the two negative allosteric modulators (MT7
and r-TIA) interact mostly with the external part of their receptor
targets while r-Da1a interacts with the orthosteric binding site.
We found one discrepancy in our study, namely that r-Da1a
shows incomplete competition with radioligands in equilibrium
binding studies, a property normally characteristic of allosteric
modulators. Our combined data, indicate that r-Da1a binds at
least in part within the orthosteric pocket of the a
1A
-AR, however
the large size of the toxin and/or its slow dissociation rate appear
to cause altered pharmacology. In yeast membranes expressing the
a
1A
-AR, both prazosin and r-Da1a cause complete displacement
of
125
I-r-Da1a, whereas like in CHO-K1 and COS-7 cells, r-
Da1a displaces only 85% of
3
H-prazosin binding [9]. Several
three-finger snake toxins display similar incomplete competition
for radioligand binding to GPCRs, however in the case of MT7
binding to the M1 muscarinic receptor, this residual binding is
readily explained by an allosteric mode of interaction [11,14,16].
r-Da1b and MTa are also unable to fully inhibit
3
H-rauwolscine
binding to a
2
-ARs despite showing no effect on the
3
H-
rauwolscine dissociation rate, but their modes of action have still
not been fully established [10,57]. A third interesting case is that of
r-TIA, which has an allosteric mode of action at a
1B
-ARs but has
been described as a competitive antagonist of the a
1A
-AR in
functional assays [19]. Despite this, r-TIA produces only 80%
inhibition of
125
I-HEAT binding in membranes from HEK-293
cells transfected with the a
1A
-AR. We initially thought that all a
1A
-
ARs present in membrane preparations may be accessible to small
molecule radioligands, but that a sub-population of the receptors
might exist in conformations that are inaccessible to the larger
toxin. This could reflect steric hindrance or a mixed allosteric/
orthosteric mode of action of r-Da1a, however if this were the
case, and the two populations of receptors were in equilibrium, the
residual binding should change over time. We found that this was
not the case, as the residual binding showed no time dependence
over 2–24 hours. Thus the two receptor pools are not inter-
changeable, suggesting possible separation between distinct
membrane compartments. This question remains to be resolved
for r-Da1a but also for other toxins that display atypical
pharmacological properties.
Key questions arising from our work are to determine which
residues of r-Da1a bind within the a
1A
-AR orthosteric site, and
whether additional regions bind to a
1A
-AR extracellular loops as
seen for MT7 and r-TIA. Identification of additional receptor
binding sites will be of interest because any such extra-orthosteric
interaction may contribute to the a
1A
-AR selectivity of r-Da1a, as
well as the insurmountable antagonism observed here in cell-based
assays, on isolated rat [9] or human muscle and in in vivo
experiments [44]. These questions will be addressed by charac-
terization of mutated r-Da1a, by determining the crystal structure
of the toxin, and by subsequent docking studies (for example [13]).
In conclusion, our findings demonstrate competitive behavior of
the r-Da1a toxin at the a
1A
-AR and highlight the crucial role of
residues located in the a
1A
-AR orthosteric site for the toxin
interaction. Thus, despite the fact that r-Da1a and MT7 belong to
the same three-finger fold structural family of toxins, and interact
with homologous biogenic amine receptors, the mode of
interaction with their respective targets is distinct. Evolution of
snake toxins has thus not only generated a wide range of
pharmacological activities from a unique peptide scaffold, but also
various strategies to interact with similar molecular targets.
Supporting Information
File S1 Recombinant expression of r-Da1a.
(DOCX)
Acknowledgments
Dr. Michael Brownstein (JCVI), Dr. Diane Perez (The Cleveland clinic
foundation, Ohio, USA) and Dr. Herve´ Paris (INSERM, U858, Toulouse,
France) for providing expression plasmids and cell lines.
Author Contributions
Conceived and designed the experiments: LB RS DH BE DS NG.
Performed the experiments: AM JM EM CR ML BG CF EL CM AL.
Analyzed the data: AM RS DH BE DS NG. Contributed reagents/
materials/analysis tools: AM BE NG. Wrote the paper: AM RS BE DS
NG.
References
1. Escoubas P, King GF (2009) Venomics as a drug discovery platform. Expert Rev
Proteomics 6: 221–224.
2. Halai R, Craik DJ (2009) Conotoxins: natural product drug leads. Nat Prod Rep
26: 526–536.
Binding Mode of an Adrenoceptor-Selective Toxin
PLOS ONE | www.plosone.org 10 July 2013 | Volume 8 | Issue 7 | e68841
3. Lewis RJ, Garcia ML (2003) Therapeutic potential of venom peptides. Nat Rev
Drug Discov 2: 790–802.
4. Shen GS, Layer RT, McCabe RT (2000) Conopeptides: From deadly venoms to
novel therapeutics. Drug Discov Today 5: 98–106.
5. Maı
¨
ga A, Mourier G, Quinton L, Rouget C, Gales C, et al. (2012) G protein-
coupled receptors, an unexploited animal toxin targets: Exploration of green
mamba venom for novel drug candidates active against adrenoceptors. Toxicon
59: 487–496.
6. Servent D, Fruchart-Gaillard C (2009) Muscarinic toxins: tools for the study of
the pharmacological and functional properties of muscarinic receptors.
J Neurochem 109: 1193–1202.
7. Sharpe IA, Thomas L, Loughnan M, Motin L, Palant E, et al. (2003) Allosteric
alpha 1-adrenoreceptor antagonism by the conopeptide rho-TIA. J Biol Chem
278: 34451–34457.
8. Rajagopalan N, Pung YF, Zhu YZ, Wong PT, Kumar PP, et al. (2007) Beta-
cardiotoxin: a new three-finger toxin from Ophiophagus hannah (king cobra)
venom with beta-blocker activity. Faseb J 21: 3685–3695.
9. Quinton L, Girard E, Maiga A, Rekik M, Lluel P, et al. (2010) Isolation and
pharmacological characterization of AdTx1, a natural peptide displaying specific
insurmountable antagonism of the alpha(1A)-adrenoceptor. Br J Pharmacol 159:
316–325.
10. Rouget C, Quinton L, Maiga A, Gales C, Masuyer G, et al. (2010) Identification
of a novel snake peptide toxin displaying high affinity and antagonist behaviour
for the alpha2-adrenoceptors. Br J Pharmacol 161: 1361–1374.
11. Fruchart-Gaillard C, Mourier G, Marquer C, Menez A, Servent D (2006)
Identification of various allosteric interaction sites on M1 muscarinic receptor
using 125I-Met35-oxidized muscarinic toxin 7. Mol Pharmacol 69: 1641–1651.
12. Fruchart-Gaillard C, Mourier G, Marquer C, Stura E, Birdsall NJ, et al. (2008)
Different interactions between MT7 toxin and the human muscarinic M1
receptor in its free and N-methylscopolamine-occupied states. Mol Pharmacol
74: 1554–1563.
13. Marquer C, Fruchart-Gaillard C, Letellier G, Marcon E, Mourier G, et al.
(2011) Structural Model of Ligand-G Protein-coupled Receptor (GPCR)
Complex Based on Experimental Double Mutant Cycle Data: MT7 SNAKE
TOXIN BOUND TO DIMERIC hM1 MUSCARINIC RECEPTOR. Journal
of Biological Chemistry 286: 31661–31675.
14. Mourier G, Dutertre S, Fruchart-Gaillard C, Menez A, Servent D (2003)
Chemical synthesis of MT1 and MT7 muscarinic toxins: critical role of Arg-34
in their interaction with M1 muscarinic receptor. Mol Pharmacol 63: 26–35.
15. Waelbroeck M, De Neef P, Domenach V, Vandermeers-Piret MC, Vanderm-
eers A (1996) Binding of the labelled muscarinic toxin 125I-MT1 to rat brain
muscarinic M1 receptors. Eur J Pharmacol 305: 187–192.
16. Olianas MC, Adem A, Karlsson E, Onali P (2004) Action of the muscarinic
toxin MT7 on agonist-bound muscarinic M1 receptors. European Journal of
Pharmacology 487: 65–72.
17. Kukkonen A (2004) Muscarinic Toxin 7 Selectivity Is Dictated by Extracellular
Receptor Loops. Journal of Biological Chemistry 279: 50923–50929.
18. Ragnarsson L, Wang CI, Andersson A, Fajarningsih D, Monks T, et al. (2013)
Conopeptide rho-TIA Defines a New Allosteric Site on the Extracellular Surface
of the alpha1B-Adrenoceptor. J Biol Chem 288: 1814–1827.
19. Lima V, Mueller A, Kamikihara SY, Raymundi V, Alewood D, et al. (2005)
Differential antagonism by conotoxin rho-TIA of contractions mediated by
distinct alpha1-adrenoceptor subtypes in rat vas deferens, spleen and aorta.
Eur J Pharmacol 508: 183–192.
20. Evans BA, Broxton N, Merlin J, Sato M, Hutchinson DS, et al. (2011)
Quantification of functional selectivity at the human alpha(1A)-adrenoceptor.
Mol Pharmacol 79: 298–307.
21. Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (K1)
and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an
enzymatic reaction. Biochem Pharmacol 22: 3099–3108.
22. Christopoulos A, Parsons AM, Lew MJ, El-Fakahany EE (1999) The assessment
of antagonist potency under conditions of transient response kinetics.
Eur J Pharmacol 382: 217–227.
23. Lew MJ, Angus JA (1995) Analysis of competitive agonist-antagonist interactions
by nonlinear regression. Trends Pharmacol Sci 16: 328–337.
24. Lew MJ, Angus JA (1997) An improved method for analysis of competitive
agonist/antagonist interactions by non-linear regression. Ann N Y Acad Sci 812:
179–181.
25. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of
spatial restraints. J Mol Biol 234: 779–815.
26. Horn F, Bettler E, Oliveira L, Campagne F, Cohen FE, et al. (2003) GPCRDB
information system for G protein-coupled receptors. Nucleic Acids Res 31: 294–
297.
27. Vroling B, Sanders M, Baakman C, Borrmann A, Verhoeven S, et al. (2011)
GPCRDB: information system for G protein-coupled receptors. Nucleic Acids
Res 39: D309–319.
28. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, et al.
(2007) High-Resolution Crystal Structure of an Engineered Human {beta}2-
Adrenergic G Protein Coupled Receptor. Science 318: 1258–1265.
29. King GF, Gentz MC, Escoubas P, Nicholson GM (2008) A rational
nomenclature for naming peptide toxins from spiders and other venomous
animals. Toxicon 52: 264–276.
30. Avlani V (2003) Application of a Kinetic Model to the Apparently Complex
Behavior of Negative and Positive Allosteric Modulators of Muscarinic
Acetylcholine Receptors. Journal of Pharmacology and Experimental Thera-
peutics 308: 1062–1072.
31. Mohr K, Trankle C, Holzgrabe U (2003) Structure/activity relationships of M2
muscarinic allosteric modulators. Receptors Channels 9: 229–240.
32. Leppik RA, Mynett A, Lazareno S, Birdsall NJ (2000) Allosteric interactions
between the antagonist prazosin and amiloride analogs at the human alpha(1A)-
adrenergic receptor. Mol Pharmacol 57: 436–445.
33. Hamaguchi N, True TA, Saussy DL Jr, Jeffs PW (1996) Phenylalanine in the
second membrane-spanning domain of alpha 1A-adrenergic receptor deter-
mines subtype selectivity of dihydropyridine antagonists. Biochemistry 35:
14312–14317.
34. Cavalli A, Fanelli F, Taddei C, De Benedetti PG, Cotecchia S (1996) Amino
acids of the alpha1B-adrenergic receptor involved in agonist binding: differences
in docking catecholamines to receptor subtypes. FEBS Lett 399: 9–13.
35. Takahashi K, Hossain M, Ahmed M, Bhuiyan MA, Ohnuki T, et al. (2007)
Asp125 and Thr130 in transmembrane domain 3 are major sites of alpha1b-
adrenergic receptor antagonist binding. Biol Pharm Bull 30: 1891–1894.
36. Ahmed M, Hossain M, Bhuiyan MA, Ishiguro M, Tanaka T, et al. (2008)
Mutational analysis of the alpha 1a-adrenergic receptor binding pocket of
antagonists by radioligand binding assay. Biol Pharm Bull 31: 598–601.
37. Waugh DJ, Zhao MM, Zuscik MJ, Perez DM (2000) Novel aromatic residues in
transmembrane domains IV and V involved in agonist binding at alpha(1a)-
adrenergic receptors. J Biol Chem 275: 11698–11705.
38. Hwa J, Perez DM (1996) The unique nature of the serine interactions for alpha
1-adrenergic receptor agonist binding and activation. J Biol Chem 271: 6322–
6327.
39. Chen S, Xu M, Lin F, Lee D, Riek P, et al. (1999) Phe310 in transmembrane VI
of the alpha1B-adrenergic receptor is a key switch residue involved in activation
and catecholamine ring aromatic bonding. J Biol Chem 274: 16320–16330.
40. Hwa J, Graham RM, Perez DM (1995) Identification of critical determinants of
alpha 1-adrenergic receptor subtype selective agonist binding. J Biol Chem 270:
23189–23195.
41. Waugh DJ, Gaivin RJ, Zuscik MJ, Gonzalez-Cabrera P, Ross SA, et al. (2001)
Phe-308 and Phe-312 in transmembrane domain 7 are major sites of alpha 1-
adrenergic receptor antagonist binding. Imidazoline agonists bind like
antagonists. J Biol Chem 276: 25366–25371.
42. Wetzel JM, Salon JA, Tamm JA, Forray C, Craig D, et al. (1996) Modeling and
mutagenesis of the human alpha 1a-adrenoceptor: orientation and function of
transmembrane helix V sidechains. Receptors Channels 4: 165–177.
43. Shim JY, Bertalovitz AC, Kendall DA (2011) Identification of essential
cannabinoid-binding domains: structural insights into early dynamic events in
receptor activation. J Biol Chem 286: 33422–33435.
44. Palea S, Maiga A, Guilloteau V, Rekik M, Guerard M, et al. (2012) Effects of
rho-Da1a a peptidic alpha1a-adrenoceptor antagonist in human isolated
prostatic adenoma and anesthetized rats. Br J Pharmacol in press.
45. Perret BG, Wagner R, Lecat S, Brillet K, Rabut G, et al. (2003) Expression of
EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells:
a potential expression system for large-scale production of G-protein coupled
receptors. Protein Expression and Purification 31: 123–132.
46. Grunewald S, Haase W, Molsberger E, Michel H, Reilender H (2004)
Production of the Human D2S Receptor in the Methylotrophic Yeast P.
pastoris? Receptors and Channels 10: 37–50.
47. Kenakin T (2005) New concepts in drug discovery: collateral efficacy and
permissive antagonism. Nat Rev Drug Discov 4: 919–927.
48. Kenakin T, Miller LJ (2010) Seven transmembrane receptors as shapeshifting
proteins: the impact of allosteric modulation and functional selectivity on new
drug discovery. Pharmacol Rev 62: 265–304.
49. Compeer MG, Meens MJ, Hackeng TM, Neugebauer WA, Holtke C, et al.
(2012) Agonist-dependent modulation of arterial endothelinA receptor function.
Br J Pharmacol 166: 1833–1845.
50. Lazareno S, Birdsall NJ (1995) Detection, quantitation, and verification of
allosteric interactions of agents with labeled and unlabeled ligands at G protein-
coupled receptors: interactions of strychnine and acetylcholine at muscarinic
receptors. Mol Pharmacol 48: 362–378.
51. Strader CD, Sigal IS, Register RB, Candelore MR, Rands E, et al. (1987)
Identification of residues required for ligand binding to the beta-adrenergic
receptor. Proc Natl Acad Sci U S A 84: 4384–4388.
52. Porter JE, Hwa J, Perez DM (1996) Activation of the alpha1b-adrenergic
receptor is initiated by disruption of an interhelical salt bridge constraint. J Biol
Chem 271: 28318–28323.
53. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, et al. (2000)
Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289: 739–
745.
54. Hanson MA, Roth CB, Jo E, Griffith MT, Scott FL, et al. (2012) Crystal
structure of a lipid G protein-coupled receptor. Science 335: 851–855.
55. Wu B, Chien EY, Mol CD, Fenalti G, Liu W, et al. (2010) Structures of the
CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists.
Science 330: 1066–1071.
56. Avlani VA, Gregory KJ, Morton CJ, Parker MW, Sexton PM, et al. (2007)
Critical role for the second extracellular loop in the binding of both orthosteric
and allosteric G protein-coupled receptor ligands. J Biol Chem 282: 25677–
25686.
57. Koivula K, Rondinelli S, Na¨ sman J (2010) The three-finger toxin MTa is a
selective a2B-adrenoceptor antagonist. Toxicon 56: 440–447.
Binding Mode of an Adrenoceptor-Selective Toxin
PLOS ONE | www.plosone.org 11 July 2013 | Volume 8 | Issue 7 | e68841
