Báo cáo khoa học: NMR structure of the thromboxane A2 receptor ligand recognition pocket pot

NMR structure of the thromboxane A
2
receptor ligand recognition
pocket
Ke-He Ruan, Jiaxin Wu, Shui-Ping So, Lori A. Jenkins and Cheng-Huai Ruan
Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, The University of Texas Health
Science Center, Houston, TX, USA
To overcome the difficulty of characterizing the structures of
the extracellular l oops (eLPs) of G protein-coupled receptors
(GPCRs) other than rhodopsin, we have explored a strategy
to generate a three-dimensional structural model for a
GPCR, the thromboxane A
2
receptor. This three-dimen-
sional structure was completed by the assembly of the NMR
structures of the computation-guided constrained peptides
that mimicked the extracellular loops and connected to the
conserved seven transmembrane domains. The NMR
structure-based model reveals the structural features of the
eLPs, in which the s econd extracellular loop (eLP
2
)andthe
disulfide bond between the first extracellular loop (eLP
1
)and
eLP
2
play a major role in forming t he ligand r ecognition
pocket. The eLP
2
conformation is dynamic a nd regulated

by the oxidation and reduction of the d isulfide bond, which
affects ligand docking in the initial recognition. The reduced
form of the thromboxane A
2
receptor experienced a decrease
in ligand b inding activity due to the rearrangement of
the e LP
2
conformation. The ligand-bound receptor was,
however, r esistant to the reduction inactivation because the
ligand covered the d isulfide bond and stabilized the e LP
2
conformation. This mole cular mechanism of ligand recog-
nition is the first that may be applied to other prostanoid
receptors and other GPCRs.
Keywords: G protein-coupled receptor; thromboxane A
2
;
thromboxane A
2
receptor; NMR; synthetic peptide.
Prostanoids, comprising prostaglandins and thromboxane
A
2
, act as local hormones in the vicinity of their production
site to regulate hemostasis and smooth muscle function.
These hormones are mediated by specific receptors inclu-
ding five basic types based on sensitivity to prostaglandin
D
2

(PGD
2
), prostaglandin E
2
(PGE
2
), prostaglandin F
2
(PGF
2
), prostaglandin I
2
(PGI
2
) and thromboxane A
2
(TXA
2
), termed DP, EP, FP, I P and TP receptors,
respectively [1,2]. In addition, EP is subdivided into four
subtypes, EP1, EP2, EP3 and EP4 receptors, based on the
responses to various agonists and antagonists. Of the
prostanoid receptors, human TP was first purified from
platelets in 1989 and the cDNA was cloned from the
placenta in 1991 [3,4]. All of the known p rostanoid receptors
belong to the rhodopsin-type G protein-coupled receptor
(GPCR) superfamily, which is one of the largest protein
families in nature with seven hydrophobic transmembrane
domains [5,6]. Because of the difficulty in crystallizing the
membrane proteins of GPCRs, rhodopsin is the only one

for which a crystal structure has been determined [7–10].
The crystal structure of rhodopsin has offered a structural
template of the conserved transmembrane helices for othe r
GPCRs, including prostanoid receptors. For more than a
decade, structural and functional studies of the prostanoid
receptors have been foc used on the identification of the
ligand binding site and specific recognition of ligands. The
homology modeling-based mutagenesis for the transmem-
brane domains of the prostanoid receptors has suggested
that the conserved regions in the third and seventh
transmembrane domains are involved in binding the
common structures o f the prostanoids, which includes a
carboxylic acid, a hydroxyl group at position 15, and two
aliphatic side chains [11–13]. To understand the different
physiopathological actions of the prostanoids, it is import-
ant to know the molecular mechanism of how the prost-
anoid receptors recognize ligand molecules selectively on the
extracellular side of the receptors, transfer them into the
membrane domains, and finally trigger the different G
proteins binding on the intracellular side of the receptors.
Structural characterization of the extracellular domains of
the r eceptors is a key step to revealing the molecular action
mechanism at the molecular level.
The crystal structure of rhodopsin cannot mimic the
extracellular domains of prostanoid receptors, such as the
TP receptor, because of the different sizes and lack o f
conservation (Fig. 1A) in the segments, which has resul-
ted in the inability to model the extracellular domains for
functional studies in general for the prostanoid receptors.
The possible involvement in ligand recognition of the

extracellular loops of prostanoid receptors and other
GPCRs has been reported by different research grou ps
(Table 1). However, the lack of an experime ntal three-
dimensional structural model for any of the receptors has
Correspondence to K H. Ruan, Division of Hematology, Department
of Internal Medicine, University of Texas Health Science Center at
Houston, 6431 Fannin St. Houston, Texas 77030, USA.
Fax: + 1 713 500 6810, Tel.: + 1 713 500 6769,
E-mail:
Abbreviations: GPCR, G protein-coupled receptor; eLP, extracellular
loop; TP, thromboxane A
2
receptor.
(Received 1 4 March 2004, revised 13 May 2004,
accepted 27 M ay 2004)
Eur. J. Biochem. 271, 3006–3016 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04232.x
impaired further definition of the ligand recognition
pocket on the extracellular side of the receptors. This
has become a major obstacle to the further understanding
of the molecular mechanism of the specific ligand
recognition in the receptors, and to the further develop-
ment of specific ligand recognition-based drugs. To
overcome this obstacle, we recently developed a strategy
to precisely mimic the extracellular l oops of the TP
receptor, termed Ôcomputation-guided constrained peptide
synthesisÕ for t he solution structural determination using
two-dimensional NMR spectroscopy. Three-dimensional
structures of eLP
2
[14] and the third extracellular l oop

(eLP
3
) [15] regions of the TP receptor have been
successfully determined by this experimental approach
individually. In addition, through the use o f the NMR
structure of the TP eLP
2
peptide, the unique residues
involved in forming the specific ligand recognition site
were identified and confirmed by NMR structure-guided
mutagenesis [1]. However, to define the specific ligand
recognition pocket of the receptor, a three-dimensional
structural model f or the t hree extracellular l oops config-
ured to the transmembrane domains is required. I n this
report, the eLP
1
structure of the TP receptor was
determined by two-dimensional N MR spectroscopy, and
the information was combined with the defined N MR
structures of eLP
2
[14] and eLP
3
[15] domains to
construct a solution structure, which includes all three
extracellular loops connected to the conserved transmem-
brane helices of the T P receptor. The NMR structure-
based extracellular loop model is the first experimental
three-dimensional structure for the prostanoid receptors
and also the first for mammalian GPCRs with the single

exception of bovine rhodopsin. As expected, the three-
dimensional model provided valuable information reveal-
ing the dynamic specific ligand recognition pocket in the
extracellular loops of the TP receptor and the ligand/
receptor recognition mechanism, which may also apply to
other prostanoid receptors.
Materials and methods
Peptide synthesis and purification
A peptide mimicking the TP eLP
1
(residues 88–104), with
homocysteine added at both e nds of the loop was s ynthes-
ized using the fluorenylmethoxycarbonyl-polyamide solid
phase method. After cleavage with trifluoroacetic acid, the
peptide was purified to homogeneity by HPLC [14]. For
cyclization of the peptide by the formation of a disulfide
bond, the purified peptide was dissolved in 1 mL dimethyl
Fig. 1. Sequence alignment of the extracellular loops of TP receptor
with rhodopsin (A) and topology model of the TP receptor (B). The
heavy line represents t he eLP
1
region studied. The amino acid sequence
of the region synthesized is shown in t he op en form and in the con-
strained loop form that has a conne ction between the N a nd C termini
by a disulfide bond using additional homocysteine (hCys) residu es.
eLP, Extracellular loop; iLP, intracellular loop; NT, N-terminal
region; CT, C-terminal region.
Table 1. Ligand recognition sites localized in extracellular loops.
Receptor Method(s) Loop Residues Reference
Prostanoid Receptors

TP Mutation eLP
2
Cys105, Cys183 [24]
EP Mutation eLP
2
Trp199, Pro200, Thr202 [30]
TP Affinity labeling eLP
2
Cys183–Asp193 [50]
TP Mutation, NMR eLP
2
Val176, Leu185
Thr186, Leu187
[1]
Other GPCRs
Thyrotropin-releasing
hormone receptor
Mutation eLP
2
Tyr181 [51]
Human P2Y
1
receptor Molecular modeling, mutation eLP
2
, eLP
3
Cys42, Cys296 [52]
A1/A3 adenosine receptor Molecular modeling,
radioligand binding assays
eLP

2
11 residues at the
C-terminal of eLP
2
[53]
Human A2a adenosine receptor Mutation eLP
2
Glu151, Glu161, Glu169 [54]
a1-Adrenergic receptor Mutation eLP
2
Gly196, Val197, Thr198 [55]
Ó FEBS 2004 Thromboxane A2 receptor structure and function (Eur. J. Biochem. 271) 3007
sulfoxide,andaddedtoH
2
O at a final concentration of
0.02 mgÆmL
)1
. The solution was then adjusted to pH 8.5
using triethylamine, and stirred overnight at room tempera-
ture. The cyclic peptide was then lyophilized and purified by
HPLC on the C4 column. The sequence of the synthesized
TP receptor eLP
1
isshowninFig.1B.
1
H NMR and assignment
Proton NMR was carried out on a Brucker 600
spectrometer in the Chemistry Department, University
of H ouston (Houston, TX). All t wo-dimensional e xperi-
ments (DQF-COSY, TOCSY and NOESY) were

performed under the same conditions (298 K, in 20 m
M
,
pH 6.0 sodium phosphate buffer containing 10% D
2
Oto
provide a lock signal). The NOESY spectrum was
recorded with a mixing time of 200 ms; the TOCSY
spectrum was carried out with a MLEV spin-lock
sequence with a total mixing time of 5 0 ms. All spectra
were composed of 2048 complex points in the F2 and 512
complex points in F1 w ith 64 s cans per t1 in crement.
Quadrature detection was achieved in the F1 by the
states-time proportional phase increment method. The
NMR data were processed using
FELIX
2000 (Accelrys
Inc., San Diego, CA). All f ree induction decays were zero-
filled to 2K-2K before Fourier transformation, and 0° (for
DQF-COSY), 70° (for TOCSY), or 90° (for NOESY)
shifted sinbell
2
window function was used in both
dimensions. The sequence-specific assignment was
obtained using the standard method [16] with the help
of the
FELIX
2000 auto-assignment program.
Calculation of structures
The overall structure of the peptide was determined through

the use of the intraresidue sequential NOEs and dihedral
angles of the peptide residues as described previously [14].
The NOE cross-peak volumes in NOESY spectrum were
converted into upper bounds of the interproton distances by
using the
FELIX
program. NOE cross-peaks were segmented
using a statistical segmentation function and c haracterized
as strong, medium and weak, corresponding to upper
bound distance range constraints of 2.5, 3 .5 and 6.0 A
˚
,
respectively. Dihedral angles were obtained by direct
measurement of
3
J
NHa
values in the DQF-COSY spectrum.
Distance geometry calculations were then carried out on an
SGI workstation using the
DGII
program within the
INSIGHT
II
package (Accelrys Inc., San Diego, CA) and initial
structures were calculated based on the NOE constraints
and dihedral angles. Energy refinement calculations, inclu-
ding restrained minimization/dynamics, were carried out in
the best distance geometry structures using the
DISCOVER

program within the
INSIGHT II
package.
Molecular modeling of the transmembrane domains
of the TP receptor
The three-dimensional structural working model for the
seven transmembrane helices of the TP receptor was
constructed u sing homology modeling within the
INSIGHT II
program, based on the bovine rhodopsin crystallographic
structure [10].
Results
Design and synthesis of peptides mimicking extracellular
loops of the human TP receptor
Our approach for the structural characterization of the TP
extracellular loops is the use of synthetic peptides mimicking
the loop domains. Analysis of the TP receptor model,
generated from molecular modeling based on the crystal-
lographic structure of bovine rhodopsin, indicated that
approximately 10–14 A
˚
separates the N and C termini of
the extracellular loops. A loop peptide whose termini are
constrained to this separation is presumably more likely to
mimic the native loop structure than the corresponding loop
peptide with unrestricted ends. In our previous studies, a
constrained p eptide corresponding to the highly co nserved
eLP
2
(residues 173–193) of the TP receptor has been made

with the N and C termini connected by a homocysteine
disulfide bond. The overall three-dimensional structure of
the loop peptide has also been determined through two-
dimensional NMR, complete
1
H NMR assignments a nd
structural construction [14]. The structure shows b-turns at
residues 180 and 185. The distance between the N and C
termini of the peptide shown in the NMR structure is
14.2 A
˚
, which corresponds to the distance (14.5 A
˚
) between
the t wo transmembrane helices connecting eLP
2
in the TP
receptor model. In addition, the constrained eLP
2
peptide
was shown to actively interact with a TP receptor ligand,
SQ29 548 which w as identified by fluorescent, CD [14], and
NMR studies [1]. The identity of the residues in contact with
the ligand, using the peptide, was further confirmed in
recombinant protein [1]. These findings suggested that the
constrained peptide approach could be used to mimic other
extracellular membrane loops of the receptor. Very recently,
based on eLP
2
peptide synthesis and structural determin-

ation, the NMR structure of a constrained peptide
mimicking the TP eLP
3
domain has been determined [15].
To further define the ligand recognition pocket in the
extracellular loops for the TP receptor, experimental three-
dimensional structure of the eLP
1
region is needed. In t his
paper, a synthetic peptide corresponding to the TP eLP
1
(Fig. 1B) was synthesized by the constrained peptide
synthesis technique using homocysteines to link t he N and
C termini of the peptide, forming a designed distance to
connect the corresponding transmembrane domains. After
synthesis and cyclization of the constrained eLP
1
peptide,
HPLC was used to purify the peptide to homogeneity. The
correct molecular mass of the peptide was then confirmed
by mass spectrometry [17].
NMR and assignment for TP eLP
1
Two-dimensional
1
H NMR spectra of the constrained eLP
1
peptide were recorded in H
2
O as described in Experimental

procedures, and the
1
H NMR assignments were a ccom-
plished by the standard sequential a ssignment t echnique
[16,18–20] as described [14]. A ll of the assignments were
performed i n a procedure including spin system identifica-
tion and sequential assignment using a combination of
TOCSY (Fig. 2A), DQF-COSY (data not shown), and
NOESY (Fig. 2B) spectra. T he complete proton reson-
ance assignments for the constrained eLP
1
peptide are
3008 K H. Ruan et al. (Eur. J. Biochem. 271) Ó FEBS 2004
summarized in Table 2. The correct assignment for the
peptide was further validated by the
FELIX
2000 auto-
assignment program.
Secondary structure of the constrained eLP
1
peptide
analyzed using two-dimensional NMR data
Using the assignment information, the secondary structures
of the peptides could be predicted by analysis of the
chemical shifts, inter-residue NOE connectivities, and the
3
J
NHa
coupling c onstants. The
3

J
NHa
coupling c onstants
were obtained by the direct measurement of
3
J
NHa
values
and by comparing the intensities of NH-aH cross peaks,
which were grouped as strong (J > 6 Hz), medium (J ¼ 4–
6 Hz) and weak (J < 4 Hz ). Weak and strong
3
J
NHa
coupling constants have been used to identify helical and
b-shee t structures, respectively [21,22]. It has been well
established that the chemical shifts, which deviate from the
Ôrandom coilÕ reference values (conformational shifts), are
closely correlated to the type of secondary structure in
proteins and peptides [16,23]. In particular, aHandNH
conformational shifts have been proposed as markers for
characterization of a peptide’s helical structures in solution.
Large conformational s hifts (> 0.3 p.p.m. upfield) are a
sensitive and powerful sign f or the presence of a helical
structure. The medium-range NOE connectivities in the
NOESY spectra and the strength of the
3
J
NHa
coupling

from the NH-CaH c ross peaks in the DQF-COSY spectra
are summarized in Fig. 3. These data suggests the presence
of a b-turn segment within peptide residues 12–15, and an
a chelix within peptide residues 2–9.
Construction of the three-dimensional structure
of the constrained eLP
1
peptide
After the complete assignment, the volumes of the identified
cross-peaks in the NOESY spectra of the peptide were
converted into c onstraints b y the
FELIX
2000 program.
Fig. 2. Assigned TOCSY and NOESY spectra
of the TP eLP
1
. (A) Expanded aH-NH
region of the T OCSY spe ctrum (50 ms mixing
time) for the T P eLP
1
in H
2
O. The spectrum
was recorded at 298 K. (B) Expanded aH-NH
region of the N OESY spectrum (200 ms
mixing time) for the TP eLP
1
in H
2
O. This

spectrum was also r ecorded at 298 K.
Ó FEBS 2004 Thromboxane A2 receptor structure and function (Eur. J. Biochem. 271) 3009
A total of 208 constraints were obtained, including 76
intraresidue, 78 s equential and 54 lon g range. The number
of the constraints p er residue for the eLP
1
peptide i s s hown
in Fig. 4. In addition, nine dihedral angles for eLP
1
were
extracted from the DQF-COSY spectrum. On the basis of
the NOE constraints and dihedral angles, first-generation
structures of the eLP
1
peptide, 100 in total, w ere obtained
using the
DGII
program. To further refine the conformation,
energy refinement calculations (minimization/dynamic)
were then carried out based on t he best distance geometry
structure using
DISCOVER
, and 12 structures were obtained
and superimposed as shown in Fig. 5. The view of the
NMR structures clearly shows the b-turn co nformations in
the residues 12–15. The distance between the N and C
termini of TP eLP
1
is 12.4 A
˚

(Fig. 5 ). The a helical structure
was localized between residues 2 and 9.
Configuration of the NMR structures of the three
extracellular loops in TP receptor model
The NMR structures of eLP
2
and e LP
3
were grafted onto
the TP receptor working m odel, which was constructed by
Table 2. Proton chemical shifts for TP eLP
1
peptide.
Residues HN Ha Hb1Hb2 Others
hCys1 4.446 2.024 2.456
Gln2 8.691 4.453 1.897 2.283
His3 8.609 4.287 3.114 3.191 7.255, 8.544
Ala4 8.155 4.252 1.280
Ala5 8.202 4.205 1.269
Leu6 7.962 4.211 1.363 1.457 0.738,
0.803,
1.457
Phe7 8.002 4.441 2.896 7.095
Glu8 7.95 4.205 1.764 1.865 2.177
Trp9 8.013 4.435 3.120 7.176,
7.376, 7.495,
10.03
His10 7.878 4.393 2.908 3.049 7.076, 8.473
Ala11 8.025 4.075 1.269
Val12 8.013 3.998 1.976 0.832

Asp13 8.424 4.883 2.638 2.841
Pro14 4.323 2.212 1.941, 3.739
Gly15 8.361 3.794, 3.875
Cys16 7.954 4.411 2.872
Arg17 8.308 4.276 1.704 1.799 1.552, 3.120,
7.132
Leu18 8.178 4.311 1.540 0.797, 0.844,
1.599
hCys19 8.237 4.464 2.005 2.471
Fig. 3. Amino acid sequence of the TP receptor eLP
1
and a survey of
sequential and medium range NOEs, HN-Ha coupling constants of the
peptide. The values of the
3
J
NHa
coupling constants a re repo rted in the
notation of S ( strong), M ( medium), and W (weak). For the sequential
NOE connectivities d
aN(i,i+1)
,d
aN(i,i+2)
,d
aN(i,i+3 )
,d
aN(i,i+4)
,d
NN(i,i+1)
,

d
NN(i,i+2)
,andd
NN(i,i+3)
are indicated by lines starting and ending a t
the position of t he intera cting residues. At t he bottom of the figure, the
location of the helix struc ture and b-turn are shown.
Fig. 4. Number of constraints per residue for TP eLP
1
. Intraresidue
(filled bars), sequential (hatched bars), and lo ng range ( open bars).
Fig. 5. Superimposition of the best 12 structures of the constrained TP
eLP
1
peptide using just the a-carbons obtained from energy refinement
calculations. The distance b etween the N and the C termini (residues
2–18) is 12.4 A
˚
.
3010 K H. Ruan et al. (Eur. J. Biochem. 271) Ó FEBS 2004
homology modeling using the crystallographic s tructure of
bovine rhodopsin as a template [10,14,15]. The configur-
ation was performed by the connection of the individ ual
loop structures to the corresponding transmembrane
domains, u sing the common trans conformation for t he
peptide bond. For eLP
1
, the detailed connections in the
three-dimensional structural view are shown in Fig. 6 .
After c onnection o f t he NMR structures o f the three

extracellular loops to the c orresponding modeled structures
of the transmembrane domains (Fig. 7A), the next key
factor in setting a correct conformation of the three loops
together was to adjust the configuration b etween the loops.
A disulfide bond was formed between residue 105 in eLP
1
andresidue183ineLP
2
(Fig. 7B); this bond has been
identified by m utagenesis [24] and protein chemical studies
[25]. After the formation of the disulfide bond, a 500-step
energy-minimization was used to refine the topological
arrangement of the three loops (Fig. 7 B). The major
conformational change o f the extracellular loops was
observed in the region with the sequence WCF in eLP
2
,
which is a highly conserved region in the prostanoid
receptors (Fig. 7). To test the extent of flexibility for the
configured topology of the three loops, a dynamic approach
was used, in which the molecular movement was stimulated
by changes in the temperature. Limited conformational
change (rmsd ¼ 1.2 A
˚
) was observed in the dynamic
studies. These results indicated t hat the configuration of
the extracellular loops was in a reasonable format (Fig. 8).
Structural characterization of the ligand recognition
site of TP receptor
Recombinant protein studies, including mutagenesis and

chimerical molecules, and molecular modeling based on
the crystal structure of rhodopsin have indicated that the
nonpeptide ligands are found mainly in deep ligand-
bound sites a mong the transmembrane domains [26–28].
However, the conserved residues in the transmembrane
Fig. 6. Detailed connectivities for the TP eLP
1
with the transmembrane region before the
connection (A), and after connection (B). The
distances between the ends of eLP
1
and
the two helices (TM1 and TM2) connecting
the loop are shown.
Fig. 7. Conformation of the assembled
extracellular l oops and the residues that form
the ligand recognition pocket of the human TP
receptor. (A) Prior to formation of disulfide
bond, and therefore prior to the formation of
the ligand recognition pocket. (B) After the
formation of the disulfide bond between
Cys105 in eLP
1
and Cys183 in eLP
2
, resulting
in the formation of th e ligand recognition
pocket. The transmembrane domains are
indicated as TMs.
Ó FEBS 2004 Thromboxane A2 receptor structure and function (Eur. J. Biochem. 271) 3011

domains could not explain the ligand selectivity in many
GPCR subfamilies, such as the prostanoid receptors. To
understand the ligand selectivities, it is important to
identify the initial contact residues in the extracellular
domains of the receptors. R ecently, the identification of
ligand recognition sites on the extracellular loops has
focused on prostanoid receptors and other GPCR
(Table 1). The three-dimensional NMR st ructural model
of the extracellular loops of the TP receptor allows a more
clear understanding of the molecular mechanism of
specific ligand recognition involving the extracellular
loops.
The NMR experimental structure of the extracellular
loops configured to the tramsmembrane domains provided
the first three-dimensional structural information about the
ligand recognition pocket of the TP receptor. The structural
information revealed s everal critical molecular mechanisms
of the specific ligand/receptor interaction, which has been
studied over a decade.
Cyclic oxidation-reduction reactions on the extracellular
loops that regulate the ligand binding affinity of the TP
receptor were reported in 1990 [29]. In the absence of
structural information about the extracellular loops of the
receptor no explanation could be offered for t he molecular
mechanism. By using our NMR three-dimensional struc-
tural model we are able to display, for the first time, the
formation of the disulfide bond between eLP
1
and eLP
2

.
The conformation of the residues in eLP
2
that form the
ligand recognition pocket is very d ynamic in the oxidation
and reduction conditions. Fig. 7 shows the conformation of
eLP
2
simulated by the presence (Fig. 7A) and absence
(Fig. 7B) of dithiothreitol, using a computational approach.
The disulfide bond between Cys105 in eLP
1
and Cys183 in
eLP
2
was reduced by breaking the bond using
BUILDER AND
BIOPOLYMER
in the
INSIGHT II
. The conformation changes of
the eLPs without the disulfide bond were studied by 500-
step energy minimization using
DISCOVER
. In a ddition, a
dynamic calculation according to changes in temperature
was also used for monitoring the conformation movement
and defining the final conformation of the eLPs. These
studies were limited to the three eLP domains. The
conformation of the eLPs in the simulated reduced form

of the TP receptor is similar to that of the conformation
before the oxidation of the disulfide bond (Fig. 7A). The
main conformational change w as localized in eLP
2
,for
which the distance of approximately 6.6 A
˚
between eLP
1
Fig. 8. Dynamic study of the configured
topology of the three loops extracellular
connected to the transmembrane domains
(TMs)oftheTPreceptor.Limited
conformational change was observed
(20 structures, rmsd 1.2 A
˚
).
3012 K H. Ruan et al. (Eur. J. Biochem. 271) Ó FEBS 2004
and eLP
2
in the oxidized form of the TP receptor was
changed to 15.0 A
˚
by reduction in the reduced form; this
resulted in a change in the diameter of the ligand recognition
pocket (Fig. 1). These structural changes of eLP
2
explain the
difference in biological activity of the receptor under
oxidation and re duction conditions. T he native receptor

with intact binding activity with its ligand should be in the
oxidized form. This structural o bservation f urther supports
that residues of Val176, Leu185, Thr186, and Leu187 in
eLP
2
region are involved in ligand recognition, which has
been concluded f rom our very current NMR a nd mutagen-
esis studies [1].
Mutation of the conserved sequence WCF in eLP
2
of the
EP receptor has been reported t o change the ligand binding
recognition of t he receptor [30]. The relationship of t he
changes of the conserved residues in eLP
2
that affected the
selectivity of the ligand recognition could be a ddressed by
structural information about the e xtracellular loops. The
possible explanation provided from our structural model is
that the changes of the conserved WCF residues in eLP
2
can
affect the dynamic conformational cha nges of eLP
2
,which
lead to the changes in the ligand docking affinity. The NMR
structural model also suggests that the conserved sequence
WCF in the eight prostanoid receptors is essential to the
formation of secondary and three-dimensional conforma-
tions for all of the ligand docking po ckets of the prostanoid

receptors. However, t he residues show no involvement in
the selectivity of ligand binding.
In the experiment carried out under at pH 7.4 and 30 °C
(similar to the physiological conditions) the ligand-dockin g
site could be protected from dithiothreitol inactivation of
the TP receptor through prior occupation with the ligand
[29]. Our NMR three-dimensional structure of the extra-
cellular loops revealed the molecular mechanism of the
docking of SQ29, 548 into the ligand recognition pocket
based on t he contacts between eLP
2
and the lig and as
defined by NMR and mutagenesis studies [1]. The initial
docking was set up by the three contacts using the
constraints between c2H of Val176 with H2 of SQ29,548,
c2H of Thr186 with H8 of SQ29 548 and d1H of Lue187
with H7 of SQ29 548 [1]. A 2000-step energy minimization
was then used to find the suitable fit of SQ29 548 in the
ligand recognition pocket. Figure 9 shows the relationship
of the disulfide bond with ligan d docking. In the free form of
the TP receptor, the exposed disulfide bond on the surface
of the molecule can be easily broken by a reducing reagent
(Fig. 9A). In c ontrast, in the ligand-bound form of the
receptor, the disulfide bond is completely covered by the
ligand, which protects the dithiothreitol reduction (Fig. 9B).
This finding confirmed the hypothesis i n which the disulfide
bond is near to the ligan d-docking site of the TP receptor
predicted by Dorn in 1990 [29] and Tai’s group in 1996 [24].
Identification of the ligand recognition pocket of the TP
receptor on the extracellular domain does not conflic t with

the ligand-binding pocket identified in TM3 and TM7. The
reason for this agreement is the ligand first coming into
contact with t he recognition site on the extracellular
domain. The second step will then be the deposition of
the ligand into the TM pocket causing the conformation
change of the receptor and triggering the coupling of the
receptor with the G protein in the intracellular domain s.
The first step of binding determines the ligand selectively
and the second step of binding is required for performance
of the receptor function. To test the hypothesis of a two-
ligand interaction site, SQ29, 548 was used to dock onto the
identified ligand recognition pocket (Fig. 10A), and the
ligand was then moved into the transmembrane binding
pocket (Fig. 10B) [12,13]. The energy calculation was
allowed to move the ligand from the recognition pocket to
the transmembrane binding pocket. The distance between
the two sites is about 23.0 A
˚
based on the NMR s tructural
model.
Discussion
Synthetic peptides have been widely used to mimic parts of
proteins in order to examine the structu re and functions of
selected portions of native proteins, particularly for mem-
brane-bound proteins, w hich are difficult to be crystallized
for X-ray studies [31–33]. Membrane proteins are generally
inserted into a bilayer during protein synthesis. Engelman
and Steitz [34] proposed that insertion of a helical hairpin
loop structure into t he membrane involves the f ormation of
a helical dimer through helix–helix interactions. Lin and

Addison’s work on the insertion of membrane helices of
integral membrane proteins suggested that the connecting
peptide forms a loop to stabilize the transmembrane helix
dimer in p reparation for membrane insertion [35]. The
primary s tructure of the c onnecting loops may contain
information su fficient to fold into native turn structures
Fig. 9. The role of the disulfide bond in relation
to ligand binding of the TP receptor. (A) The
ligand bind ing poc ke t i s o pen and the disulfi de
bond is exposed. (B) S Q29, 548 is bound to
the ligand binding pocket, therefore conceal-
ing the disulfide bond.
Ó FEBS 2004 Thromboxane A2 receptor structure and function (Eur. J. Biochem. 271) 3013
which have biological activities even in the absence of the
flanking transmembrane helices. Both Takemoto et al.[36]
and Konig et al. [37] f ound that isolated peptides compri-
sing the C-terminal domain, or the second or third loops in
the intracellular portions of bovine rhodopsin themselves
have biological activity. These results with the prototypical
G protein-coupled protein suggest that extramembraneous
parts of the GPCRs may independently fold into a native
structure. However, the free peptide in solution may not
necessarily adopt an ordered conformation, especially for
the t erminal residues, which a re particularly important for
the configuration of a peptide mimicking a loop structure
and connected to other defined structures. This led us to
develop an approach of computation-guided constrained
peptide synthesis for structural and functional studies of the
GPCRs.
In most cases, structural studies of the extracellular

domains and ligand recognition sites of mammalian GPCRs
have been performed b y the homology modeling approach
using the crystal structure of rhodopsin as a template
[25,38–44]. Due to lower conservation of the extrameme-
brane domains between rhodopsin and mammalian
GPCRs, especially in the case of prostanoid receptors
(Fig. 1), little information about the structural characteris-
tics of the extracellular domains of the prostanoid receptors
is available. Crystal structures for the prostanoid receptors
are unlikely to be obtained in the near future. Assembly of
the NMR structures of the extracellular d omains connected
to the transmembrane domain described above for the TP
receptor offers an alternative way to quickly characterize the
structural features of the extracellular loops of mammalian
GPCRs. The principle of this strategy includes the steps of
the computer-guided constrained peptide synthesis with
precise secondary structural configurations, two-dimen-
sional NMR structural determinations, and the fragment
structural configuration on the transmembrane domains.
The homocysteine disulfide bond used to constrain t he
secondary structure of the synthetic peptides, mimicking the
TP extracellular loops, can be applied to other GPCRs a s it
is believed that the seven t ransmembrane domains of most
of the mammalian GPCRs share similar conformations and
separations between the h elices connecting the loops. In
addition, the constrained peptide synthesis approach also
provided an opportunity to synthesize peptides that mim-
icked the intracellular loops for t he TP and other GPCRs
for structural and functional studies. It should be noted that
Yeagle et al. determined the synthetic p eptides with free

ends that mimicked the intracellular loops of bovine
rhodopsin and provided t hree-dimensional s tructures f or
the loops in which the peptides adopt a turn conformation
[45–49]. But the defined distance between both of the ends of
the peptide were not conclusive because the structures of the
N- and C-terminal residues of p eptides were varied. Our
constrained peptide overcomes this problem because the
constrained N- and C-terminal residues are considered as
intraresidues and adopted a conformation similar to that of
other residues in solution. The NMR structures described
above have confirmed this hypothesis in which the c on-
strained peptides gave a defined conformation for the
terminal residues of the TP extracellular loops.
The identified ligand recognition pocket, located mainly
between eLP
1
and eLP
2
of the TP receptor, and the residues
important for contacting the ligand in e LP
2
have been
supported by mutagenesis studies [24,30] and an affinity
labeling experiment [50] for the prostanoid receptors
reported from different groups. We could not exclude the
possibility that the eLP
1
region may also contain residues
involved in forming the ligand recognition pocket. How-
ever, based on our findings and affinity labeling studies, it

can be concluded t hat the ligand is a nchored mainly to the
TP eLP
2
region. This information h as offered a structural
template to predict the specific ligand recognition pockets in
the same location for other prostanoid receptors. This
prediction is based o n the following facts of the eight
prostanoid receptors: first, all of the receptors share s imilar
topological backbones and the eLP
2
s are highly co nserved;
second, the cysteine residues making up the disulfide bond
between eLP
1
and eLP
2
are also conserved; third, muta-
genesis for the residues in the eLP
2
region for t he EP3
receptors showed the effect of the ligand binding selectivities
[30]; and lastly, t he affinity-labeling s tudies for t he TP
receptor showed that the initial ligand binding site is in eLP
2
[50]. Our finding is also in agreement with the current
observation for the human P2Y
1
receptor, a mammalian
Fig. 10. Docking of the TP receptor with its
ligand. (A) SQ29 548 docking onto the iden-

tified ligand recognition pocket. The docking
was performed in respect to the contacts be-
tween SQ29 5 48 with the residues V176, L185,
T186 and L187 in eLP
2
identified previously
[1].(B) SQ29 548 at the TM binding pocket .
The docking was based on the c ontac ts
between SQ29 548 with S201 and R295 as
described previously [ 12,13]. The distance
SQ29 548 moved from the ligand recognition
pocket to the TM pocket was calculated to be
 23.0 A
˚
.
3014 K H. Ruan et al. (Eur. J. Biochem. 271) Ó FEBS 2004
GPCR, in which the ligand recognition site (Ômate-binding
siteÕ) is localized on the extracellular domain and the
Ôprincipal TM binding siteÕ is in the TM domains [25]. By
homology alignment of the eLP
2
regions of the eight
prostanoid r eceptors, our NMR structural m odel of the
ligand recognition pocket in the TP receptor f urther implies
that the conserved residues in the eLP
2
s and the disulfide
bond configuration maintain general ligand recognition
pockets, and that the variable residues within the eLP
2

of
the prostanoid receptors play key roles in the specific ligand
recognition which determines the affinity between the
receptor and ligand. This hypothesis can be used to explain
the observation that each prostanoid receptor can cross-
react with other prostanoids with the only difference being
in the binding affinity. Determination of the three-dimen-
sional structural conformation of the extracellular loops and
experimental identification of the ligand recognition pockets
for other prostanoid receptors will provide evidence to test
our predictions.
Acknowledgements
We thank Dr X. Gao, Chemistry Department, University of Houston,
for acc ess to the NMR facility. An acknowledgement is also made to
the Robert A. Welch Foundation and the W. M. Keck Center for
Computational Biology at the University of Houston for computer
resource support. This work was s upported by NIH Grants HL56712
and NS23327. The NMR facility at University of Houston is founded
by the W. M . Keck Foundation.
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