![The murine sequence (top) corresponds to that whose structure is presented in the crystal structure of the mouse mu opioid receptor. The helical secondary structure assigned with Stride
[56] is shown as yellow rectangles. The gray residues in between TM5 and TM6 (MLSGSK) are absent in the crystal structure. The helical secondary structure of the wsMUR-TM model assigned with Stride is shown as blue lines. (B) Superposition of the mouse mu opioid receptor (yellow) and the wsMUR-TM model (blue). (C) Rendering from the “extracellular” viewpoint of the crystal structure of mouse mu opioid receptor, where the side chain of the mutated positions in wsMUR are depicted as blue spheres. The majority of mutations (50 out of 55) are located at the exterior of the structure. Five remaining positions (in green, see also green squares in Figure. 2A) are also rendered: Y130, T120, A306, N232, and K305. None of these positions are in direct contact with the irreversible antagonist β-FNA based on the crystal structure [15], where β-FNA was covalently attached to K235.](https://www.researchgate.net/profile/Felipe-Matsunaga-2/publication/242015095/figure/fig2/AS:202751087779843@1425351034081/The-murine-sequence-top-corresponds-to-that-whose-structure-is-presented-in-the-crystal_Q320.jpg)
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A Computationally Designed Water-Soluble
Variant of a G-Protein-Coupled Receptor: 'e
Human Mu Opioid Receptor
Jose Manuel Perez Aguilar
University of Pennsylvania9.;.C*0><*<>9.77.->
Jin Xi
Felipe Matsunaga
Xu Cui
Bernard Selling
See next page for additional authors
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A Computationally Designed Water-Soluble Variant of a G-Protein-
Coupled Receptor: 'e Human Mu Opioid Receptor
Abstract
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A Computationally Designed Water-Soluble Variant of a
G-Protein-Coupled Receptor: The Human Mu Opioid
Receptor
Jose Manuel Perez-Aguilar
1.¤
,JinXi
2.
, Felipe Matsunaga
2
,XuCui
2,3
, Bernard Selling
4
, Jeffery G. Saven
1
*,
Renyu Liu
2
*
1Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2Department of Anesthesiology and Critical Care, University
of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 3Department of Anesthesiology, Beijing Tongren Hospital, Capital Medical University, Beijing,
China, 4Impact Biologicals Inc., Swarthmore, Pennsylvania, United States of America
Abstract
G-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by
pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large
quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants
of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties.
Here we present the first study involving the computational design, expression and characterization of water-soluble variant
of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of
the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure
was highly similar to the subsequently determined structure of the murine receptor and was used to computationally
design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous
media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural
and functionally related features with the native human MUR, including helical secondary structure and comparable affinity
for the antagonist naltrexone (K
d
= 65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor
variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble
variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.
Citation: Perez-Aguilar JM, Xi J, Matsunaga F, Cui X, Selling B, et al. (2013) A Computationally Designed Water-Soluble Variant of a G-Protein-Coupled Receptor:
The Human Mu Opioid Receptor. PLoS ONE 8(6): e66009. doi:10.1371/journal.pone.0066009
Editor: Yang Zhang, University of Michigan, United States of America
Received February 21, 2013; Accepted April 30, 2013; Published June 14, 2013
Copyright: ß2013 Perez-Aguilar 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: This work was supported by the Foundation for Anesthesia Education and Research (FAER) (PI, RL), National Institutes of Health (NIH) K08 (K08-GM-
093115-01) (PI, RL), GROFF (PI, RL), the Department of Anesthesiology and Critical Care at the University of Pennsylvania (PI, RL), and the Penn Nano/Bio Interface
Center through the National Science Foundation (NSF) NSEC DMR-0425780 (PI, JGS). NSF MRSEC DMR-1120901 (JGS) provided infrastructural support related to
protein computational work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors of this manuscript declare that there is no competing interest among authors. Dr. Bernard Selling is the president of Impact
Biologicals. Impact Biologicals is not involved in any projects that will financially benefit (or be harmed) by publication of this work. Including Dr. Bernard Selling
as one of the coauthors does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: liur@uphs.upenn.edu (RL); saven@sas.upenn.edu (JGS)
¤ Current address: Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, United States of America
.These authors contributed equally to this work.
Introduction
The G-protein-coupled receptor (GPCR) family of proteins
have important roles in signal transduction and cellular response
to extracellular stimuli [1] and are the targets of many
pharmaceuticals. Drug development and the study of the
molecular mechanisms of (GPCRs) are impeded by limited
solubility and difficulty in isolating sufficient quantities of
functional receptors. These difficulties are caused in part by the
large numbers of hydrophobic residues on the transmembrane,
lipid-contacting protein exterior. To circumvent these problems,
water-soluble variants of GPCRs can potentially be identified by
systematically redesigning these exterior residues. Along these
lines, computational protein design has been used to create water-
soluble analogs of transmembrane proteins that can be expressed
in E. coli and that retain structural and functional features of their
parent membrane proteins [2,3], e.g., the bacterial potassium
channel (KcsA) [4] and a transmembrane domain of the nicotinic
acetylcholine receptor (nAChR) [5]. Herein, such design is
extended to a member of the GPCR superfamily, where
comparative modeling is used to identify exterior residues in the
transmembrane region.
The mu opioid receptor (MUR) is a GPCR that is the dominant
target of opioids, many of which are potent analgesics widely used
for the treatment of severe and chronic pain, e.g., morphine [6].
Opioid use has soared in recent years [7–9], and human MUR has
been linked to many of its notorious side effects, including
addiction and deadly respiratory depression [6,7]. The molecular
mechanisms governing GPCR function remain obscure despite
the profound insights obtained recently from multiple high-
resolution crystal structures [10–18].
PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e66009
Here computational redesign to increase water solubility while
retaining functionally related properties was applied to the human
MUR. In previous redesign efforts, template structures were
derived from experimental structures of KcsA (via X-ray
diffraction) [4,19] and nAChR (via cryo-electron microscopy)
[5,20]. Often with membrane proteins (including GPCRs), such
experimentally determined structures are not available. No
structure for the human MUR was available when this study
was initiated, thus the approach was extended to include structural
modeling. The design involved several key steps: (i) Comparative
modeling using sequence alignment and known GPCR structures
(the subsequently solved structure of murine MUR provided a
means to assess the quality of the comparative model [15]); (ii)
Identification and co mputational redesign of transmembrane
exterior residues; (iii) Overexpression in E. coli and purification; (iv)
Characterization of structural and ligand-binding properties in
aqueous buffer. The designed water-soluble human MUR has
structurally and functionally related properties comparable to the
native membrane-soluble human MUR.
Materials and Methods
Comparative Modeling
Bovine rhodopsin (UniProtKB accession number P35372; PDB
accession code: 1U19) [11] and the b
2
adrenergic receptor
(UniProtKB: P02699; PDB: 2RH1) [12] were used as templates in
the creation of models of the human MUR transmembrane
domain (UniProtKB: P07550) [21]. Pairwise sequence alignments
of the human mu opioid receptor with bovine rhodopsin and with
the b
2
adrenergic receptor were carried out using BLASTp [22]
with the Blosum62 substitution matrix [23]. In a multiple sequence
alignment, adjustments were performed to maintain highly
conserved residues of the class A GPCR family [24]. One
hundred independent models of a protein structure comprising
residues S66 to C353 were generated using Modeller 8v2 [25].
The structure was validated using molprobity [26] and then
validated a posteriori by the recently solved crystal structure of the
murine mu opioid receptor [15].
Computational Protein Design
Within the comparative model structure, residues targeted for
mutation were those having more than 40% solvent exposure
(1.4 A
˚probe radius and percent exposure measured relative to
GXG tripeptide) [27] and were within previously estimated
membrane boundaries [28]. To identify the site-specific amino
acid probabilities of the target positions, a statistical entropy-based
formalism was used [4,29,30]. In this theoretical approach, an
effective entropy, which was a function of the site-specific
probabilities of the amino acids and their conformational states,
was maximized by varying the probabilities subject to energetic
constraints on the sequences using a Lagrange multiplier method.
The set of probabilities corresponding to the optimum was used to
guide protein redesign. Energy functions to quantify sequence-
structure compatibility were derived from a molecular mechanics
force field [31]. To account for solvation effects and for the
tendency of different amino acids to be exposed to or sequestered
from water (hydrophobicity), an effective energy (herein environ-
mental energy) was employed that was based on the local density
of C
b
atoms of each residue and parameterized using a database of
soluble, globular proteins [4,30]. In this case the environmental
energy term was constrained to a value expected for soluble
proteins having 288 residues [4,30], the size of the TM domain of
the human MUR [4]. The conformational variability of the amino
acid residues was addressed using a rotamer library of side chain
conformations [32]. The site-specific probabilities of the amino
acids at each of the variable positions were determined by
maximizing an effective entropy function subject to constraints on
the two energies. These probabilities were used to identify specific
sequences. After the residues targeted for potential mutations were
identified, the remaining residues were fixed at their wild type
identities, and their side chain conformations were allowed to vary
to accommodate possible mutations. All amino acids but proline
and cysteine were permitted at each of the identified variable
positions. Calculations proceeded as described previously [4].
Probabilities used were those for which the Lagrange multiplier b
conjugate to the average molecular force field energy took on a
value of b
21
= 0.5 kcal/mol. Identification of sequence proceeded
iteratively until amino acid identities were specified at each of the
targeted residues.
Protein Expression and Purification
The synthetic cDNA encoding of the transmembrane-only
water-soluble MUR variant (wsMUR-TM) was produced by
DNA2.0 Inc. (Menlo Park, CA). The sequences were subcloned
between the NdeI and XhoI restriction sites of the expression
plasmid pET-28b(+) (EMD/Novagen). E. coli BL21(DE3) cells
(EMD/Novagen) were used for expression. Cells were grown in
shake flasks with Lysogeny broth medium with 30 mg/mL
kanamycin to an OD of 1.0, induced with 1 mM Isopropyl b-D-
1-thiogalactopyranoside (IPTG) for 3 h at 37uC, then pelleted by
centrifugation. Cell pellets were stored at 20uC until purification.
For solubility testing, 1 OD aliquots of cells were pelleted in
microcentrifuge tubes, suspended in 150 mL of TE (50 mM Tris-
HCl, 1 mM EDTA, pH = 8.0), then shaken with 0.3 g of glass
beads (0.1 mm diameter) for 5 min. Aliquots of the resulting
lysates were spun in a microcentrifuge for 1 min. Aliquots of total
lysate, or the supernatant and pellet fractions after centrifugation,
were analyzed on reducing sodium dodecyl sulfate (SDS) gels.
Frozen cells from 250 mL of fermentation (500–550 ODs) were
thawed, and then suspended in 33.5 mL of 50 mM Tris-HCl, 1 M
urea, pH = 8.0. Once the pellet was fully resuspended, EDTA was
added to 1 mM, Triton X-100 to 1%, and hen egg lysozyme to
1mg per OD of cells, in a total volume of 37 mL. After the slurry
was incubated for 20 min at room temperature (RT), MgCl
2
was
added to 3 mM, followed by 100 units of benzonase. The
suspension was swirled, incubated another 5 min at RT, and then
spun in an Oak Ridge tube at 10,000 rpm for 20 min at 20uCin
an SS-34 rotor (r
avg
= 6.98 cm, r
max
= 10.70 cm).
The resulting pellet was resuspended into 35 mL of 50 mM
Tris-HCl, 1 M urea, pH = 8.0. Triton X-100 (1.5 mL of a 25%
solution) and 2-mercaptoethanol (2-ME) was added to 40 mM.
The tube was inverted several times, and then spun as above.
The following steps were designed to resemble those that had
been used to dissolve and purify recombinant forms of native mu
opioid receptor. The pellet from the above washes was
resuspended into 5 mL of buffer phosphate Tris buffer (100 mM
phosphate, 10 mM Tris, adjusted to pH = 8.0 with NaOH) and
dispersed by drawing through a pipet followed by a 25 gauge
needle. The volume was then raised to 37 mL by addition of
phosphate Tris buffer, and 2-ME was then added to 40 mM. The
tube was inverted to mix, then spun as above.
The resulting pellet was dispersed into 36 mL of PT as
described above. The suspension was then mixed with an equal
volume of phosphate Tris buffer containing 0.2% SDS and
10 mM 2-ME. The suspension was rocked until it became almost
clear (60–90 min). The suspension was then poured into two
38 mL Oak Ridge tubes. These were spun tube at 12,000 rpm for
20 mins at 20uC in an SS-34 rotor.
A Water-Soluble Variant of the Human Mu Receptor
PLOS ONE | www.plosone.org 2 June 2013 | Volume 8 | Issue 6 | e66009
Mass Spectrometry
The appropriate protein band from an SDS-PAGE gel was
excised and digested with trypsin. Peptides were injected into a
nano-LC/MS (10 cm C18 capillary column) to be separated by
Eksigent. NanoLC proteomics experiments were run at 200 nL/
min for 60 min with gradient elution. Nanospray was used to
spray the separated peptides into LTQ (Thermo Fisher Scientific,
MA). The raw data was acquired by Xcalibur (Xcalibur, Inc.
Arlington). Sequest (http://fields.scripps.edu/sequest/) was used
to search the database Uniprot_Sprot, Scaffold 2.6 (Proteome
Software, Inc. OR) was used to combine and analyze the Sequest
generated data quantitatively by using spectrum count.
Circular Dichroism and Thermal Stability
Circular dichroism (CD) spectra were recorded by using CD
Spectrometer (Chirascan, AppliedPhotophysics Limited, Leather-
head, United Kingdom) with a scan speed of 1 nm/s and 1 mm
path length. Corresponding blanks were used for calibration for
each assay and subtracted from raw data. Two data sets were
recorded and averaged to increase the signal-to-noise ratio. The
CDNN CD spectra deconvolution software [33] was utilized to
determine the secondary structure content of the proteins. CD
spectroscopy for wsMUR-TM at different temperatures were
recorded with 6 mM of the receptor in buffer (5 mM sodium
phosphate, pH = 7.0) from 10uCto90uC in increments of 2uC per
min. Absorbance was maintained lower than 1.0 to ensure
sufficient light transmission. The temperature-dependence curve
was plotted using GraphPad Prism (version 5, GraphPad Software,
Inc. La Jolla).
Protein Unfolding and Thermal Stability
The CD spectra of the protein was determined in the presence
and absence of 8 M urea with and without 2-ME (25 mM or
200 mM, 5 mM sodium phosphate, 0.01% SDS, pH = 7.0). The
final samples contained protein diluted to 6 mM and the requisite
dilutions of urea and 2-ME. Each sample was incubated at room
temperature for 1 h.
CD spectroscopy was utilized to investigate the thermostabiliza-
tion of wsMUR-TM by cholesterol. The CD spectra were
recorded with a 6 mM of the receptor (0.27 mg/ml) in buffer
(5 mM sodium phosphate, 0.01% SDS, pH = 7.0) from 10uCto
90uC. 0.01% SDS was used since cholesterol can be dissolved in
SDS solution. Cholesterol with 1:1 molar ratio to the protein was
used. The thermostability determination protocol is similar as
described above.
Protein unfolding was also studied by monitoring the intrinsic
tryptophan fluorescence of the protein. A RF-5301PC spectro-
fluorophotomer (Shimadzu North America, Columbia, MD) was
used to monitor fluorescence emission following excitation at
295 nm. Samples were prepared as in CD unfolding experiments.
Samples were measured in duplicate and results reflect averaged
values of each trial.
Homogeneous Time-Resolved Fluorescence (HTRF)
Based Binding Assay
The fluorescent binding assay employs the native MUR fused at
the N-terminus to a SNAP-tagHenzyme and expressed on
HEK293 cells. SNAP-tag-mu-opioid is then covalently labeled
with terbium cryptate (Lumi4H-Tb), a long lifetime FRET donor.
An analog of the potent opioid antagonist naltrexone that contains
the d2 dye (red-naltrexone) is used as the fluorescence energy
transfer acceptor. Upon ligand binding, a FRET process occurs
between the Lumi4-Tb donor (emission at 620 nm) in SNAP-
Lumi4-Tb-mu-opioid receptor and the red-naltrexone acceptor
(emission at 665 nm). The fluorescence emission from the acceptor
is detected in a time resolved manner (TR-FRET). For HTRF
assay (Cisbio Bioassays, Bedford, MA), Tag-liteHmu opioid cells
suspended in culture medium were dispensed in white 384-well
low-volume microplates (Greiner Bio-one Greiner Bio-One North
America, Monroe, NC) at 3700 cells/10 mL/well Tag-lite m
opioid labeled cells with 60 nM of Tag-lite opioid receptors red-
naltrexone, and 5 mL of the wsMUR-TM with 11 further 1:2
serial dilutions from the mM to the nM range. All samples were
mixed with final volume 20 ml and incubated at RT for 2 h. After
incubation, HTRF signals were measured using a plate reader
(BMG, Cary, NC) after excitation at 337 nm at both 620 and
665 nm emission, HTRF signal was calculated as a two-
wavelength signal ratio: [intensity (665 nm)/intensity (620 nm)].
IC
50
determination and statistical analysis IC
50
values for
wsMUR-TM were determined by fitting the dose–responses
curves using the Prism program (GraphPad Software, San Diego,
CA).
Results
Computational Design of a Water-Soluble Variant of the
Human MUR
A comparative model of the human MUR transmembrane
domain (288 residues, comprising sites 66–353) was obtained using
known GPCR structures [25] (Figure 1). Based upon surface
accessibility, 55 exterior transmembrane residues were selected for
the computational redesign. As described in previous work, the
calculations identify the probabilities of amino acids at variable
positions among sequences that are expected to be water soluble
by imposing overall energetic constraints such that (a) the amino
acids are consistent with the remainder of the protein in terms of
sterics, electrostatics and hydrogen-bonding (as guided by a
molecular force field) and (b) a solvation or environmental energy
is constrained to have a value expected for a globular protein of
the same length, yielding exterior, hydrophilic residues expected
for a water-soluble protein [4,5]. A first calculation identified 31 of
the targeted positions as having a strong preference for one amino
acid, i.e., those sites where the probability one amino acid
exceeded 0.8: A75E
1.37
, S78K
1.40
, I79K
1.41
, V83E
1.45
, F89K
1.51
,
Y93E
1.55
, T120E
2.54
, K187K
4.43
, I188E
4.44
, V191E
4.47
,
C192K
4.48
, A199E
4.55
, L202K
4.58
, M205E
4.61
, N232D
5.36
,
L233K
5.37
, I240K
5.44
, F241K
5.45
, I244E
5.48
, M245E
5.49
,
L248K
5.52
, V252E
5.56
, A289E
6.42
, V293K
6.46
, P297E
6.50
,
I300K
6.53
, I303K
6.56
, I304E
6.57
, A306K
6.59
, L326K
7.41
, and
V336K
7.51
. The superscript notation is consistent with the
Ballesteros and Weinstein indexing system: (number of the
transmembrane helix).(residue number relative to most conserved
residue in transmembrane helix, which is assigned position 50)
[34]. These mutations were introduced, and the corresponding
residue identities were fixed in subsequent calculations. Similarly,
second and third calculations respectively specified one (V82E
1.44
)
and two (T72K
1.34
and L333E
7.48
) additional mutations. From
the results of a fourth calculation, the most probable amino acid
was selected at the remaining 21 positions, yielding a sequence and
model structure for wsMUR-TM as presented in Figure 1. The
designed sequence is presented in figure 2A.
The recent structure of the closely related murine MUR
provides an opportunity to evaluate the structure and the location
of the mutated positions in wsMUR-TM [15]. The human and
mouse receptors have 94% sequence identity. The model of the
human MUR and the murine crystal structure superimpose well
(Figure 2B), particularly with regard to the transmembrane helices
A Water-Soluble Variant of the Human Mu Receptor
PLOS ONE | www.plosone.org 3 June 2013 | Volume 8 | Issue 6 | e66009
[35]. Only five positions in wsMUR-TM were not located in the
exterior of the murine structure (T120E, Y130K, N232D, K305G,
and A306K) and could in principle affect ligand binding
(Figure 2C). In the murine structure, however, these five positions
residues were not among the residues that directly contact beta-
Funaltrexamine (b-FNA), an irreversible antagonist of the receptor
[15].
Overexpression, Purification, and Verification of wsMUR-
TM
Attempts to express the native full-length human MUR in E. coli
were unsuccessful presumably due to the protein’s toxicity. In
contrast, wsMUR-TM expressed well and was isolated with high
purity using affinity chromatography (Figure 3A). The yield was
,20 mg/L of shake flask culture. An initial exposure to ,0.1%
sodium dodecyl sulfate (SDS) was required to purify the receptors.
After dialysis to remove non-bound SDS, the purified variant was
soluble at 6 mg/mL in buffer solution (130 mM NaCl, 20 mM
NaHPO
4
, pH = 7.0). Using mass spectrometry (MS), fragments of
the wsMUR were identified covering 37.6% of the designed
sequences. One of the identified fragments of the receptor is
presented in Figure 3B.
Secondary Structure of wsMUR-TM
The secondary structure of the water-soluble variant was
determined through circular dichroism (CD). The CD spectra
indicated predominantly helical structures with a helical secondary
structure content of ,48% (estimations based on the molar
ellipticity over the range 205 to 260 nm). The comparison of the
helical content with that of the native human MUR expressed in
yeast system in the presence of high concentration of detergent
(0.1% SDS) is presented in table 1.
Thermal Stability, Cholesterol Interaction, and Conserved
Disulfide Bond
As monitored by CD, wsMUR-TM started to lose ellipticity
significantly near 62uC and was almost fully unfolded at 90uC
(Figure 4). The stability of wsMUR-TM was also investigated
Figure 1. Scheme of the computational design protocol. Step 1, Comparative modeling: Starting from the sequence alignment between known
GPCR structures (bovine rhodopsin and b
2
adrenergic receptor) and human MUR, a model structure of the human MUR was generated. Step 2,
Identification of exposed sites in the transmembrane portion: Using the comparative model, the transmembrane lipid-exposed positions were identified
(pink). Step 3, Computational design of selected exterior positions to generate a water-soluble variant: The selected exterior positions are targeted of the
computational redesign with the intention of increasing the protein’s solubility and ability to be overexpressed in E. coli. Residues are colored by
amino acid types: hydrophilic in green (GNQSTY); hydrophobic in white (ACFILMPVW); basic in blue (HKR); and acidic in red (DE).
doi:10.1371/journal.pone.0066009.g001
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Figure 2. (A) Sequences of the crystal structure of the mouse mu opioid receptor (PDB code 4DKL; top) (1) and the human water-
soluble variant wsMUR-TM (bottom). The murine sequence (top) corresponds to that whose structure is presented in the crystal structure of the
mouse mu opioid receptor. The helical secondary structure assigned with Stride [56] is shown as yellow rectangles. The gray residues in between TM5
and TM6 (MLSGSK) are absent in the crystal structure. The helical secondary structure of the wsMUR-TM model assigned with Stride is shown as blue
lines. (B) Superposition of the mouse mu opioid receptor (yellow) and the wsMUR-TM model (blue). (C) Rendering from the ‘‘extracellular’’ viewpoint
of the crystal structure of mouse mu opioid receptor, where the side chain of the mutated positions in wsMUR are depicted as blue spheres. The
A Water-Soluble Variant of the Human Mu Receptor
PLOS ONE | www.plosone.org 5 June 2013 | Volume 8 | Issue 6 | e66009
upon addition of cholesterol, which has been found to modulate
the stability of several GPCRs [14,36]. The inclusion of cholesterol
caused a shift of the melting point from 82.9uC to 89.3uC,
suggesting that it may stabilize the helical structure of wsMUR-
TM (Figure 5) [14,36].
CD and intrinsic tryptophan fluorescence were used to probe
disulfide bond formation [37] in the water-soluble variant. The
structure of wsMUR-TM was monitored with increasing concen-
trations of urea and the reducing agent 2-mercaptoethanol (2-
ME). After addition of urea, the molar ellipticity at 222 nm and
the intensity of the intrinsic tryptophan fluorescence of wsMUR-
TM decreased. Even in 8 M urea, the protein retains some helical
structure (Table 2). Upon addition of 2-ME, both the molar
ellipticity and fluorescence further decreased, becoming more
pronounced at the higher concentration of the reducing agent
(200 mM). Thus the presence of an intramolecular disulfide bond
is corroborated in the case of wsMUR-TM.
wsMUR Binding Assay
Naltrexone binding was monitored using a competitive TR-
FRET (time-resolved fluorescence resonance energy transfer)
based assay with fluorescently labeled wild type MUR and a
naltrexone-derived antagonist. The ratio of fluorescence emission
at 665 nm and 620 nm decreased in a dose-dependent manner
with increasing concentrations of wsMUR-TM. The determined
K
d
values for naltrexone were 6561.8 nM (wsMUR-TM)
(Figure 6). As a negative control, human serum albumin (HSA,
a soluble helical protein), rather than a water-soluble variant, was
introduced with no significant change in the fluorescence ratio
upon HSA addition.
Discussion
Computational Design of wsMUR-TM
For many membrane proteins, the local environments of
residues in the interior of the protein are similar to those observed
in the interiors of globular proteins [38–41]. For such proteins, the
exterior lipid-contacting residues, which are predominantly
hydrophobic, could in principle be redesigned so as to yield a
more water-soluble variant. This requires reliable structural
information to determine which residues are on the exterior of
the protein in the transmembrane region. Computational methods
have been used to guide such redesign. The transmembrane
domains of Streptomyces lividans (KcsA) [4,42] and the a1 subunit of
the nicotinic acetylcholine receptor (nAChR) from Torpedo
marmorata [5] have been redesigned resulting in mutations to
30% and 18% of the respective proteins [4,5,43]. The water-
soluble proteins retain structural and functional features of the
parent membrane proteins. In these studies, experimentally
derived structural information guided the selection of exterior
residues and the computational redesign at these selected sites.
Herein, the computational redesign approach was extended to
design functional water-soluble variant of a human GPCR without
explicit a priori experimental structural information (when the
effort was initiated). The GPCR system studied here is the largest
protein yet targeted for solubilization via redesign (53 out of 288
residues, approximately 18% of the protein). The human MUR
was selected due to its pharmacological relevance to understanding
pain management and opioid addiction. wsMUR-TM was
expressed in large quantities in a heterologous bacterial system
and displayed structural and functional characteristics comparable
with those of the native receptor.
The computationally guided redesign requires a structural
model of the protein. Two aspects of the redesign process are
expected to be sensitive to the accuracy of the model structure: the
identification of exterior, transmembrane positions that are
targeted for redesign and the amino acid probabilities used to
determine the redesigned sequence. Given the similarity of known
GPCR structures and the quality of the stuctures that can be
obtained with comparative modeling, of these two aspects we
expect the amino acid probabilities to be more sensitive to the
detailed structural features of a given model structure. The recent
structure of the closely related murine MUR provides an
opportunity to evaluate the quality of the modeled structure and
the location of the mutated positions in wsMUR-TM [15]. The
human and mouse receptors have 94% sequence identity. The
comparative model of the human MUR and the murine crystal
structure superimpose well, particularly in the transmembrane
region [35]. However, five targeted positions in wsMUR-TM were
not located in the exterior of the murine structure (T120E,
Y130K, N232D, K305G, and A306K). These five positions
residues were not among the eleven that directly contact the ligand
in the crystal structure; these residues compose the ‘‘binding site’’
and are those having an atom within 4 A
˚of the b-FNA ligand
present in the crystal structure [15]. Thus, the binding properties
of the wsMUR-TM are expected to be comparable with those of
the native mu opioid receptor as demonstrated in this study.
Although additional binding sites could potentially be introduced
via computational redesign of the protein, we would not expect
these to have affinities comparable to the wild type protein. In
future work, details of the binding site of the wsMUR-TM can
potentially be resolved using high-resolution structural approaches
like NMR and x-ray crystallography.
majority of mutations (50 out of 55) are located at the exterior of the structure. Five remaining positions (in green, see also green squares in Figure.
2A) are also rendered: Y130, T120, A306, N232, and K305. None of these positions are in direct contact with the irreversible antagonist b-FNA based
on the crystal structure [15], where b-FNA was covalently attached to K235.
doi:10.1371/journal.pone.0066009.g002
Figure 3. Overexpression and verification of wsMUR-TM. (A) A
SDS-PAGE gel for wsMUR-TM is shown where lane 1 correspond to the
standard, lane 2 to purified wsMUR-TM and lane 3 to expressed wsMUR-
TM in the crude material. The band corresponding to the wsMUR-TM is
indicated by a red arrow at 36 kDa. (B) Representative mass
spectrometry data for fingerprinting of an identified peptide fragment
is displayed (TATNIYIFNLAK; from the IC1-TM2 region).
doi:10.1371/journal.pone.0066009.g003
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GPCR Expression in E. coli and Protein Purification
The expression of several GPCRs in E. coli has been achieved
[44], but obtaining functional GPCRs in large quantities has
generally been challenging. While functional human MUR has
previously been successfully expressed in E. coli [45], the protein
appeared as part of a fusion construct and was obtained at a low
yield and remained unpurified. To our knowledge, successful
induction of useful amounts of native MUR without a fusion
partner (or with a His-tag) in E. coli has not been reported. The
apparently toxic effects of the human MUR to the cells may
explain the lack of such reports. Such toxicity was also observed in
this study in attempts to express the native human MUR with a
His-tag. However, production of His-tagged computationally
designed human MUR variant (wsMUR-TM) was achieved.
Thus, the toxicity of the native receptor appears to arise from
hydrophobic residues located on the exterior surface of the
receptor’s transmembrane region. The ability to express and
purify large amounts of functional GPCRs from E. coli should
greatly accelerate studies of the structure-function relationships for
such receptors.
Since an initial exposure to 0.1% SDS was required during
purification, the purified wsMUR-TM in solution may still contain
small amounts of SDS due to the difficulty of removing SDS from
proteins. In order to avoid protein aggregation, 0.01% of SDS was
utilized in the final buffer solutions for functional assays. Using
binding and crystallographic studies, we have shown that such
small amounts of SDS do not disrupt the tertiary structure and/or
the ligand binding capabilities of some proteins [46]. Conversely,
much higher concentration of SDS (0.1%) and other anionic
detergents are required for the ‘‘solubilization’’ of the native
human MUR [47].
Protein Structure Characterization and Thermostability
Consistent with the secondary structure of the native human
MUR expressed in yeast [48,49], the CD spectra of the wsMUR-
TM displayed a predominantly helical structure (48%) which is
comparable with the native full length MUR [48]. Lower
percentage of the helical content in the native full length MUR
in the literature may be due to the inclusion of the N and C
terminus and the higher concentration of the detergent (0.1%
SDS).
Intrinsic tryptophan fluorescence was used to provide qualita-
tive information of the conformations adopted by the water-
soluble receptors; wsMUR-TM contains just six tryptophan
residues (W135
2.69
, W194
4.50
, W228
EC2
, W230
EC2
, W295
6.48
,
and W320
7.35
). Of particular interest are the tryptophan residues
located in the partially buried transmembrane locations of the
model structure (underlined above). The fluorescence associated
with these residues is expected to be sensitive to the local
hydrophobic environment and overall folding of the protein. The
observed decrease in the tryptophan fluorescence and the red shift
in the emission with increasing denaturant (urea) concentration
Table 1. Helical content comparison for the native and engineered receptors.
205–260 nm wsMUR-TM (pH 7.0 in NaHPO
4
) Native MUR (pH 7.0+0.1% SDS)
Helix 48.0% 40.6%
Turn 14.6% 18.9%
Others 37.4% 40.5%
wsMUR-TM: transmembrane-only water-soluble human mu receptor variant;
MUR: human mu receptor.
doi:10.1371/journal.pone.0066009.t001
Figure 4. Mean residue ellipticity at 222 nm of wsMUR-TM in
buffer solution (5 mM sodium phosphate, pH= 7.0) as a
function of temperature, from 10 to 90uC. The spectrum of
wsMUR-TM showed significant change near 62uC and an almost
complete loss in molar ellipticity at 90uC.
doi:10.1371/journal.pone.0066009.g004
Figure 5. Molar circular dichroism (CD) derived percentage of
the original helical content (determined at 222 nm) of wsMUR-
TM in the absence (black dots) and the presence (red dots) of
cholesterol in buffer solution (5 mM sodium phosphate, 0.01%
SDS, pH = 7.0) as functions of the temperature. The addition of
cholesterol stabilized the wsMUR-TM as indicated by the rightward shift
of the thermostability curve.
doi:10.1371/journal.pone.0066009.g005
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suggest that at least some of these tryptophan residues are located
in the interior of the protein.
The decrease of the tryptophan fluorescence under denaturing
conditions and in the presence of 2-ME is consistent with the
changes in CD spectra observed under similar conditions. The
requirement of the reducing agent to fully denature and unfold the
protein indicates the relevance of an intramolecular disulfide bond
in stabilizing the receptor structure. Although these observations
suggest the presence of a disulfide bond, they do not specify which
bond is formed given the existence of 11 cysteine residues in
wsMUR-TM. However, the CD and ligand-binding studies are
consistent with the adoption of the proper protein tertiary
structure and by extension with the formation of the correct
disulfide bond.
With the exception of rhodopsin, GPCRs are not generally
stable and this represents one of the major obstacles for structural
studies [10]. Many strategies have been employed to overcome this
problem, such as the use of stabilizing ligands [50], stabilizing
mutations [51], and high salt concentrations in solution [13]. In
the present study, wsMUR-TM that is both soluble in aqueous
media and thermally stable was successfully generated by
redesigning the protein. The thermostability of wsMUR-TM
improved significantly in the presence of cholesterol. These results
are consistent with recent observations that the introduction of
cholesterol hemisuccinate increases the thermostability of the A
2A
adenosine receptor in detergent micelles [14]. Studies have
demonstrated that interactions between cholesterol and GPCRs
can play an important role in modulating the structural stability as
well as the function of the receptors [36,52]. Moreover, cholesterol
is present in the recently solved crystal structure of the murine mu
opioid receptor [15]. Two mechanisms have been proposed
regarding cholesterol and membrane proteins: a) direct interaction
between the receptor and the cholesterol molecule [36] and/or b)
a modification of the membrane microenvironment of the protein
by cholesterol [53]. Given that the experiments here were
performed in aqueous solution, the first mechanism appears to
apply to the interactions with wsMUR-TM. One of the
disadvantages of designing a water-soluble variant of an integral
protein is the inability to study the mechanism of the membrane
microenvironment modification given the absence of a membrane.
Despite this limitation, it is noteworthy that the mutations of the
lipid-contacting positions do not preclude the well-known inter-
actions that exist between the native receptor and relevant
molecules such as cholesterol. The finding that the wsMUR-TM
thermostability increased in the presence of cholesterol together
with the previous finding that the water-soluble a1 subunit of the
nicotinic acetylcholine receptor (nAChR) recovers the protein-
lipid interactions of the native receptor [5,54], supports the power
and flexibility of the solubilization approach in maintaining similar
interactions as those seen in the native membrane-soluble protein.
Ligand Binding Properties of the wsMUR-TM
A recently developed methodology which uses a fluorescently
labeled ligand and the native MUR [55] was used to investigate
the ligand-binding capabilities of the water-soluble receptors. This
binding assay has been applied to study several GPCRs and
particularly to MUR, where the K
i
values for the morphinan
opioids naloxone and naltrindole were estimated (5.1 nM and
8.1 nM for naloxone and naltrindole, respectively) and found to be
in agreement with values obtained using other techniques [55],
wsMUR-TM competes with native MUR expressed in HEK293
cells for the potent opioid antagonist naltrexone. This study clearly
demonstrates that the wsMUR-TM can compete with the native
MUR for the fluorescent antagonist with binding affinities in nM
range. The HSA (negative control) results indicate that the
interaction of the water-soluble variant with naltrexone is selective
and specific.
Conclusions and Future Directions
The findings reported in this study open new tools for the
characterization of GPCRs and the human MUR. The compu-
tational design approach could be applied more broadly to other
receptors in the GPCR superfamily, particularly those whose
structure may not be known a priori, via the use of comparative
Table 2. Effects of denaturant and reducing agent on the wsMUR-TM.
None
Urea
(8 M) Urea (8 M) 2-ME (25 mM) Urea (8 M) 2-ME (200 mM)
Molar Ellipticity (%; 222 nm) 100.0 40.0 25.1 0.0
Fluorescence Peak Intensity (%; 300–350 nm) 100.0 28.4 23.9 4.5
wsMUR-TM: transmembrane-only water-soluble human mu receptor variant;
Values are normalized to the condition without denaturant or reducing agent (None). 2-ME: 2-mercaptoethanol.
doi:10.1371/journal.pone.0066009.t002
Figure 6. Binding competition assay between the human mu
opioid receptor expressed in HEK293 cells and the mopioid
water-soluble variants. Inhibition of the native mu opioid receptor
constitutive signal in the presence of increasing concentrations of
wsMUR-FL (black dots, IC
50
= 8.4610
27
M, R
2
= 0.9306) or wsMUR-TM
(red squares, IC
50
= 8.6610
27
M, R
2
= 0.9067) in sodium phosphate
buffer. Data for the negative control is also included, HSA (inverted
green triangles). Data is used to calcualte HTRF ratios, and represent the
mean 6standard error of mean of quadruplicates. DF is used for the
comparison of different runs of the same assay which reflects the signal
to background of the assay. DF = [(Ratio
sample
-Ratio
backgroud
)/Ratio
back-
groud
](%).
doi:10.1371/journal.pone.0066009.g006
A Water-Soluble Variant of the Human Mu Receptor
PLOS ONE | www.plosone.org 8 June 2013 | Volume 8 | Issue 6 | e66009
(homology) modeling. The wsMUR-TM over-expressed in the E.
coli system would be amenable for NMR spectroscopy and other
structural studies in the presence and absence of opioid ligands.
The water solubility of the receptor and its high yield in a bacterial
production system offer great advantages for further pharmaco-
logical characterization as well as drug refinement and discovery.
Acknowledgments
The authors appreciate the outstanding technical support from Dr. Fang
Xie, Dr. Min Li at the Department of Anesthesiology and Critical Care at
the University of Pennsylvania. Dr. Renyu Liu thanks the mentorship and
valuable discussions from Dr. Roderic G. Eckenhoff and the support from
the Department of Anesthesiology and Critical Care at the University of
Pennsylvania.
Author Contributions
Conceived and designed the experiments: JGS RL. Performed the
experiments: JMP-A JX FM XC BS JGS RL. Analyzed the data: JMP-A
JX FM XC BS JGS RL. Contributed reagents/materials/analysis tools:
JGS RL. Wrote the paper: JMP-A JX FM XC BS JGS RL.
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