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
Trang 1NMR structure of the thromboxane A2 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 loops (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 A2 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 second extracellular loop (eLP2) and the
disulfide bond between the first extracellular loop (eLP1) and
eLP2 play a major role in forming the ligand recognition
pocket The eLP2conformation is dynamic and regulated
by the oxidation and reduction of the disulfide bond, which affects ligand docking in the initial recognition The reduced form of the thromboxane A2receptor experienced a decrease
in ligand binding activity due to the rearrangement of the eLP2 conformation The ligand-bound receptor was, however, resistant to the reduction inactivation because the ligand covered the disulfide bond and stabilized the eLP2 conformation This molecular 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 A2; thromboxane A2receptor; NMR; synthetic peptide
Prostanoids, comprising prostaglandins and thromboxane
A2, 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
D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2
(PGF2), prostaglandin I2 (PGI2) and thromboxane A2
(TXA2), termed DP, EP, FP, IP 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 prostanoid 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 other GPCRs, including prostanoid receptors For more than a decade, structural and functional studies of the prostanoid receptors have been focused 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 of 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 receptors 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 of 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 groups (Table 1) However, the lack of an experimental 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: kruan@uth.tmc.edu
Abbreviations: GPCR, G protein-coupled receptor; eLP, extracellular
loop; TP, thromboxane A 2 receptor.
(Received 14 March 2004, revised 13 May 2004,
accepted 27 May 2004)
Trang 2impaired 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 loops of the TP
receptor, termed computation-guided constrained peptide synthesis for the solution structural determination using two-dimensional NMR spectroscopy Three-dimensional structures of eLP2 [14] and the third extracellular loop (eLP3) [15] regions of the TP receptor have been successfully determined by this experimental approach individually In addition, through the use of the NMR structure of the TP eLP2 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 for the three extracellular loops config-ured to the transmembrane domains is required In this report, the eLP1 structure of the TP receptor was determined by two-dimensional NMR spectroscopy, and the information was combined with the defined NMR structures of eLP2 [14] and eLP3 [15] domains to construct a solution structure, which includes all three extracellular loops connected to the conserved transmem-brane helices of the TP 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 eLP1(residues 88–104), with homocysteine added at both ends of the loop was synthes-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 the eLP 1 region studied The amino acid sequence
of the region synthesized is shown in the open form and in the
con-strained loop form that has a connection between the N and C termini
by a disulfide bond using additional homocysteine (hCys) residues.
eLP, Extracellular loop; iLP, intracellular loop; NT, N-terminal
region; CT, C-terminal region.
Table 1 Ligand recognition sites localized in extracellular loops.
Prostanoid Receptors
Thr186, Leu187
[1] Other GPCRs
Thyrotropin-releasing
hormone receptor
radioligand binding assays
C-terminal of eLP 2
[53]
Trang 3sulfoxide, and added to H2O 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 eLP1is shown in Fig 1B
1
H NMR and assignment
Proton NMR was carried out on a Brucker 600
spectrometer in the Chemistry Department, University
of Houston (Houston, TX) All two-dimensional
experi-ments (DQF-COSY, TOCSY and NOESY) were
performed under the same conditions (298 K, in 20 mM,
pH 6.0 sodium phosphate buffer containing 10% D2O to
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 50 ms All spectra
were composed of 2048 complex points in the F2 and 512
complex points in F1 with 64 scans per t1 increment
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 free induction decays were
zero-filled to 2K-2K before Fourier transformation, and 0° (for
DQF-COSY), 70° (for TOCSY), or 90° (for NOESY)
shifted sinbell2 window function was used in both
dimensions The sequence-specific assignment was
obtained using the standard method [16] with the help
of theFELIX2000 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 theFELIXprogram NOE cross-peaks were segmented
using a statistical segmentation function and characterized
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 of3JNHavalues in the DQF-COSY spectrum
Distance geometry calculations were then carried out on an
SGI workstation using theDGIIprogram within theINSIGHT
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 theINSIGHT IIpackage
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 using homology modeling within theINSIGHT 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 peptide corresponding to the highly conserved eLP2(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 1H NMR assignments and 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 two transmembrane helices connecting eLP2in the TP receptor model In addition, the constrained eLP2peptide was shown to actively interact with a TP receptor ligand, SQ29 548 which was 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 eLP2peptide synthesis and structural determin-ation, the NMR structure of a constrained peptide mimicking the TP eLP3domain 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 eLP1region is needed In this paper, a synthetic peptide corresponding to the TP eLP1 (Fig 1B) was synthesized by the constrained peptide synthesis technique using homocysteines to link the N and
C termini of the peptide, forming a designed distance to connect the corresponding transmembrane domains After synthesis and cyclization of the constrained eLP1 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 eLP1 Two-dimensional1H NMR spectra of the constrained eLP1 peptide were recorded in H2O as described in Experimental procedures, and the 1H NMR assignments were accom-plished by the standard sequential assignment technique [16,18–20] as described [14] All of the assignments were performed in a procedure including spin system identifica-tion and sequential assignment using a combinaidentifica-tion of TOCSY (Fig 2A), DQF-COSY (data not shown), and NOESY (Fig 2B) spectra The complete proton reson-ance assignments for the constrained eLP peptide are
Trang 4summarized in Table 2 The correct assignment for the
peptide was further validated by the FELIX 2000
auto-assignment program
Secondary structure of the constrained eLP1peptide
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
3JNHa coupling constants The 3JNHa coupling constants
were obtained by the direct measurement of3JNHavalues
and by comparing the intensities of NH-aH cross peaks,
which were grouped as strong (J > 6 Hz), medium (J¼ 4–
6 Hz) and weak (J < 4 Hz) Weak and strong 3JNHa
coupling constants have been used to identify helical and
b-sheet structures, respectively [21,22] It has been well
established that the chemical shifts, which deviate from the
random coil reference values (conformational shifts), are
closely correlated to the type of secondary structure in proteins and peptides [16,23] In particular, aH and NH conformational shifts have been proposed as markers for characterization of a peptide’s helical structures in solution Large conformational shifts (> 0.3 p.p.m upfield) are a sensitive and powerful sign for the presence of a helical structure The medium-range NOE connectivities in the NOESY spectra and the strength of the 3JNHa coupling from the NH-CaH cross 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 eLP1peptide After the complete assignment, the volumes of the identified cross-peaks in the NOESY spectra of the peptide were converted into constraints by the 2000 program
Fig 2 Assigned TOCSY and NOESY spectra
of the TP eLP 1 (A) Expanded aH-NH
region of the TOCSY spectrum (50 ms mixing
time) for the TP eLP 1 in H 2 O The spectrum
was recorded at 298 K (B) Expanded aH-NH
region of the NOESY spectrum (200 ms
mixing time) for the TP eLP 1 in H 2 O This
spectrum was also recorded at 298 K.
Trang 5A total of 208 constraints were obtained, including 76
intraresidue, 78 sequential and 54 long range The number
of the constraints per residue for the eLP peptide is shown
in Fig 4 In addition, nine dihedral angles for eLP1were extracted from the DQF-COSY spectrum On the basis of the NOE constraints and dihedral angles, first-generation structures of the eLP1peptide, 100 in total, were obtained using theDGIIprogram To further refine the conformation, energy refinement calculations (minimization/dynamic) were then carried out based on the best distance geometry structure usingDISCOVER, and 12 structures were obtained and superimposed as shown in Fig 5 The view of the NMR structures clearly shows the b-turn conformations in the residues 12–15 The distance between the N and C termini of TP eLP1is 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 eLP2and eLP3were grafted onto the TP receptor working model, which was constructed by
Table 2 Proton chemical shifts for TP eLP 1 peptide.
0.803, 1.457
7.376, 7.495, 10.03
7.132
1.599
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 the3J NHa coupling constants are reported 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) , and d NN(i,i+3) are indicated by lines starting and ending at
the position of the interacting residues At the bottom of the figure, the
location of the helix structure and b-turn are shown.
Fig 4 Number of constraints per residue for TP eLP 1 Intraresidue (filled bars), sequential (hatched bars), and long 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 between the N and the C termini (residues 2–18) is 12.4 A˚.
Trang 6homology modeling using the crystallographic structure of
bovine rhodopsin as a template [10,14,15] The
configur-ation was performed by the connection of the individual
loop structures to the corresponding transmembrane
domains, using the common trans conformation for the
peptide bond For eLP1, the detailed connections in the
three-dimensional structural view are shown in Fig 6
After connection of the NMR structures of the three
extracellular loops to the corresponding 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 between the loops
A disulfide bond was formed between residue 105 in eLP1
and residue 183 in eLP2 (Fig 7B); this bond has been
identified by mutagenesis [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 7B) The major
conformational change of the extracellular loops was
observed in the region with the sequence WCF in eLP2, 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 (rmsd¼ 1.2 A˚) was observed in the dynamic studies These results indicated that 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 among 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 loops 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 the ligand recognition
pocket The transmembrane domains are
indicated as TMs.
Trang 7domains 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 Recently, the identification of
ligand recognition sites on the extracellular loops has
focused on prostanoid receptors and other GPCR
(Table 1) The three-dimensional NMR structural 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 several 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 the 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 eLP1 and eLP2 The conformation of the residues in eLP2 that form the ligand recognition pocket is very dynamic in the oxidation and reduction conditions Fig 7 shows the conformation of eLP2 simulated by the presence (Fig 7A) and absence (Fig 7B) of dithiothreitol, using a computational approach The disulfide bond between Cys105 in eLP1and Cys183 in eLP2was reduced by breaking the bond usingBUILDER AND BIOPOLYMERin theINSIGHT II The conformation changes of the eLPs without the disulfide bond were studied by 500-step energy minimization using DISCOVER In addition, 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 was localized in eLP2, for which the distance of approximately 6.6 A˚ between eLP
Fig 8 Dynamic study of the configured topology of the three loops extracellular connected to the transmembrane domains (TMs) of the TP receptor Limited conformational change was observed (20 structures, rmsd 1.2 A˚).
Trang 8and eLP2 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 eLP2explain the
difference in biological activity of the receptor under
oxidation and reduction conditions The native receptor
with intact binding activity with its ligand should be in the
oxidized form This structural observation further supports
that residues of Val176, Leu185, Thr186, and Leu187 in
eLP2region are involved in ligand recognition, which has
been concluded from our very current NMR and
mutagen-esis studies [1]
Mutation of the conserved sequence WCF in eLP2of the
EP receptor has been reported to change the ligand binding
recognition of the receptor [30] The relationship of the
changes of the conserved residues in eLP2that affected the
selectivity of the ligand recognition could be addressed by
structural information about the extracellular loops The
possible explanation provided from our structural model is
that the changes of the conserved WCF residues in eLP2can
affect the dynamic conformational changes of eLP2, 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 pockets of the prostanoid
receptors However, the 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-docking
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 the contacts between eLP2 and the ligand 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 ligand 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 contrast, 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 in which the disulfide bond is near to the ligand-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 conflict with the ligand-binding pocket identified in TM3 and TM7 The reason for this agreement is the ligand first coming into contact with the 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 domains 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 structural model
Discussion
Synthetic peptides have been widely used to mimic parts of proteins in order to examine the structure and functions of selected portions of native proteins, particularly for mem-brane-bound proteins, which 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 the membrane involves the formation 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 preparation for membrane insertion [35] The primary structure of the connecting loops may contain information sufficient 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 binding pocket is open and the disulfide
bond is exposed (B) SQ29, 548 is bound to
the ligand binding pocket, therefore
conceal-ing the disulfide bond.
Trang 9which have biological activities even in the absence of the
flanking transmembrane helices Both Takemoto et al [36]
and Konig et al [37] found 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 terminal residues, which are 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 by 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 domains 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 the
secondary structure of the synthetic peptides, mimicking the
TP extracellular loops, can be applied to other GPCRs as it
is believed that the seven transmembrane domains of most
of the mammalian GPCRs share similar conformations and
separations between the helices connecting the loops In
addition, the constrained peptide synthesis approach also provided an opportunity to synthesize peptides that mim-icked the intracellular loops for the TP and other GPCRs for structural and functional studies It should be noted that Yeagle et al determined the synthetic peptides with free ends that mimicked the intracellular loops of bovine rhodopsin and provided three-dimensional structures for 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 peptides 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 con-strained peptides gave a defined conformation for the terminal residues of the TP extracellular loops
The identified ligand recognition pocket, located mainly between eLP1and eLP2of the TP receptor, and the residues important for contacting the ligand in eLP2 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 eLP1region 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 that the ligand is anchored mainly to the
TP eLP2region This information has offered a structural template to predict the specific ligand recognition pockets in the same location for other prostanoid receptors This prediction is based on the following facts of the eight prostanoid receptors: first, all of the receptors share similar topological backbones and the eLP2s are highly conserved; second, the cysteine residues making up the disulfide bond between eLP1 and eLP2 are also conserved; third, muta-genesis for the residues in the eLP2 region for the EP3 receptors showed the effect of the ligand binding selectivities [30]; and lastly, the affinity-labeling studies for the TP receptor showed that the initial ligand binding site is in eLP2 [50] Our finding is also in agreement with the current observation for the human P2Y 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 548 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 contacts 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˚.
Trang 10GPCR, in which the ligand recognition site (mate-binding
site) is localized on the extracellular domain and the
principal TM binding site is in the TM domains [25] By
homology alignment of the eLP2 regions of the eight
prostanoid receptors, our NMR structural model of the
ligand recognition pocket in the TP receptor further implies
that the conserved residues in the eLP2s and the disulfide
bond configuration maintain general ligand recognition
pockets, and that the variable residues within the eLP2of
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 access 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 supported 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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