HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Salminen, T. Articles by Johnson, M. S. Search for Related Content PubMed PubMed Citation Articles by Salminen, T. Articles by Johnson, M. S. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 33, 23405-23413, August 13, 1999

Three-dimensional Models of _2A-Adrenergic Receptor Complexes Provide a Structural Explanation for Ligand Binding^*

Tiina Salminen^§¶, Minna Varis^§, Tommi Nyrönen^**, Marjo Pihlavisto, Anna-Marja Hoffrén^§§, Tuomas Lönnberg^§§, Anne Marjamäki^¶, Heini Frang, Juha-Matti Savola^§§, Mika Scheinin, and Mark S. Johnson

From the  Department of Biochemistry and Pharmacy, Åbo Akademi University and Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistökatu 6 A, FIN-20520 Turku, ^** Center for Scientific Computing (CSC) Tietotie 6, FIN-02101 Espoo, and  Department of Pharmacology and Clinical Pharmacology, Medicity, University of Turku and ^§§ Juvantia Pharma Ltd, Tykistökatu 6 A, FIN-20520 Turku, Finland

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have compared bacteriorhodopsin-based (_2A-AR_BR) and rhodopsin-based (_2A-AR_R) models of the human _2A-adrenengic receptor (_2A-AR) using both docking simulations and experimental receptor alkylation studies with chloroethylclonidine and 2-aminoethyl methanethiosulfonate hydrobromide. The results indicate that the _2A-AR_R model provides a better explanation for ligand binding than does our _2A-AR_BR model. Thus, we have made an extensive analysis of ligand binding to _2A-AR_R and engineered mutant receptors using clonidine, para-aminoclonidine, oxymetazoline, 5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304), and norepinephrine as ligands. The representative docked ligand conformation was chosen using extensive docking simulations coupled with the identification of favorable interaction sites for chemical groups in the receptor. These ligand-protein complex studies provide a rational explanation at the atomic level for the experimentally observed binding affinities of each of these ligands to the _2A-adrenergic receptor.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

₂-Adrenergic receptors (₂-ARs)¹ belong to the family of G-protein-coupled receptors (GPCRs), which are an important class of membrane proteins having an integral role in cellular signaling. Thus, GPCRs are key targets for pharmaceutical development, and ₂-ARs, in particular, have therapeutic applications in a variety of diseases, for example in hypertension, pain, depression, anxiety, and obesity.

Three-dimensional structural details are commonly used to interpret ligand binding, biochemical responses, molecular biological data, and to design rationally novel ligands as potential pharmaceutical lead compounds. Unfortunately, no atomic level structure exists for any of the adrenergic receptors. Nonetheless, sequence analysis has predicted the presence of seven transmembrane helices (TM), now universally accepted to be present in all GPCRs. A low resolution structure of bacteriorhodopsin, a proton pump in Halobacterium halobium, from cryo-electron microscopy (1) has been available for some time and has been commonly used as a template for building "homology" models of GPCRs, although it is not a GPCR. The other commonly used template for GPCR modeling includes the low resolution cryo-electron microscopy structure of bovine rhodopsin (2), which is a true representative of the GPCR family.

Recently, two new structures have become available and are currently the best representative structures of the 7-TM GPCRs. These are the 2.5-Å resolution crystal structure of bacteriorhodopsin (3), including loop regions, and the refined electron microscopy structure of frog rhodopsin (4). The frog rhodopsin structural data has been combined with the analysis of about 500 GPCR sequences to provide a template structure for the 7-TM receptor family, which is thought to be more representative of mammalian GPCRs than the bacteriorhodopsin structure (5).

The predicted helices of the 7 TM in ₂-ARs form a water-accessible binding site for ligands in a pocket or crevice between the helices in the interior of the receptor (6). Residues within this cavity directly participate in ligand binding, which leads to alteration of the receptor structure with subsequent activation of downstream signaling (7, 8). Recently, we have combined targeted mutagenesis experiments with structural modeling to show that two molecules that covalently bind to _2A-AR, chloroethylclonidine (CEC) and 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA), recognize two different receptor conformations (9). Here, we show that our most recent model, based on the vertebrate rhodopsin template (5), provides a better explanation for CEC and MTSEA binding that does another model constructed by us using the 2.5-Å bacteriorhodopsin structure (3). Furthermore, we have made an extensive analysis of ligand binding to our model based on the vertebrate rhodopsin template using clonidine, para-aminoclonidine, oxymetazoline, 5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304), and norepinephrine as ligands. The experimental ligand binding results for the recombinant receptor and engineered mutants agree well with the modes of binding observed from the modeled receptor-ligand complexes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [³H]RX821002 ([³H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline) was obtained from Amersham Pharmacia Biotech (specific activity, 56 Ci/mmol). ()-Norepinephrine was obtained from Merck. CEC, clonidine, oxymetazoline, para-aminoclonidine, phentolamine, and UK14,304 were supplied by Research Biochemicals (Natick, MA). MTSEA was purchased from Toronto Research Chemicals Inc. (North York, Canada). Cell culture reagents were supplied by Life Technologies, Inc.

Mutagenesis and Expression Vectors-- Site-directed mutagenesis was performed utilizing the Altered Sites II in vitro mutagenesis system (Promega, Madison, WI) as described previously (10). The wild type _2A-AR (11) and the mutated receptor cDNAs were subcloned into the KpnI/BamHI sites of the expression vector pREP4 (InVitrogen, NV Leek, The Netherlands).

Cell Culture and Transfections-- Adherent Chinese hamster ovary cells (American Type Culture Collection, Manassas, VA) were cultured as reported previously (10). The pREP4-based expression constructs were transfected into cells using the Lipofectin reagent kit (Life Technologies, Inc.) (10). Hygromycin B (Roche Molecular Biochemicals)-resistant (550 µg/ml) cell cultures were examined for their ability to bind the a₂-AR antagonist [³H]RX821002. The transfected cells chosen for further experiments were subsequently maintained in 200 µg/ml hygromycin B.

Reactions with CEC and MTSEA-- The second-order rate constant (k) for the reaction of CEC or MTSEA with wild type _2A-AR and each investigated mutant (_2A-ARSer201, _2A-ARSer201Cys197, _2A-ARSer201Cys200, _2A-ARSer201Cys202, _2A-ARSer201Cys203, _2A-ARSer201Cys204) was estimated by determining the extent of the reaction after a fixed time, 15 min (CEC) or 2 min (MTSEA), with 7 concentrations of CEC (0.5, 1, 5, 10, 50, 100, and 500 µM) or MTSEA (5, 20, 50, 100, 150, 200, and 250 µM) as previously reported (9, 12).

Competition Binding Assays-- Competition binding assays were performed in 50 mM K⁺-phosphate buffer (pH 7.4 at 21 °C) as described previously (10, 13). With the wild type _2A-AR, _2A-ARSer201 and _2A-ARSer201Cys197, the inhibition constants (K_i) for each competitor (clonidine, para-aminoclonidine, oxymetazoline, UK14,304, and norepinephrine) were analyzed with GraphPad Prism multicurve data analysis (GraphPad Software, San Diego, CA) for the three separate experiments performed in triplicate. For _2A-ARSer201Cys200 and _2A-ARSer201Cys204 only, the inhibition constants (K_i) were also determined for UK14,304 and norepinephrine.

Modeling and Comparison of the Models-- Models of _2A-AR ((14) SWISS-PROT accession number P08913) were built using both the high resolution x-ray crystal structure of bacteriorhodopsin ((3); Protein Data Base file code 1AP9) and a C-atom template of the transmembrane helices of the rhodopsin-like GPCRs (5) as structural templates. These models are referred to as _2A-AR_BR and _2A-AR_R, respectively. The GPCR C-atom template structure is based on the sequence comparison of about 500 different GPCR sequences and the cryo-electron microscopy structure of frog rhodopsin (5). Five different _2A-AR_BR models and five _2A-AR_R models were made using the modeling program MODELLER (15). A representative model from each set, used for all subsequent studies, was chosen after examination of the models using the program InsightII (Molecular Simulations Inc., San Diego, CA) and by choosing the model in each set with the lowest value of the objective function, which describes the degree of fit of the model to the input structural data used in its construction, derived by the program MODELLER (15).

The models were then optimally superimposed using VERTAA.² This program rapidly produces an objective comparison of two structures without any human intervention (i.e. without providing an initial alignment to seed the comparison). The program HelixTip (17) was used to define the differences in the two superimposed models, _2A-AR_BR and _2A-AR_R, by specifying the vector along each helix and then calculating the angle between the vectors of the equivalent helices in the two matched structures (i.e. the difference in the helix tilt angle of the equivalent helices).

Receptor models for each mutant were generated by replacing the corresponding residue at positions 197, 200, or 204, one at the time, with a cysteine residue and Cys-201 (the wild type sequence) with a serine residue (9).

GRID Maps-- The computer program GRID Version 16 (18) was used to map essential interactions in the binding site of each receptor model. GRID calculates energies of interaction between a probe and the receptor. The probes (Table I) were placed at different positions throughout the ligand binding site, and the receptor side chains were allowed to move too (using the side chain flexibility option in GRID). The GRID maps were visualized using the program CERIUS 2 (Molecular Simulations Inc., San Diego, CA).

Docking Simulations-- CEC and MTSEA were docked manually to the binding cavity of the _2A-AR_BR and the _2A-AR_R models as described in Marjamäki et al. (9).

Clonidine, para-aminoclonidine, oxymetazoline, UK14,304, and norepinephrine were initially energy minimized with the MM+ (extended MM2) forcefield in vacuum using the conjugate gradient method. The conformational space available to these small molecules was explored using simulated annealing and the MM+ forcefield implemented within the program Hyperchem 5.01 (Hypercube Inc., Gainesville, FL). Simulations were carried out in a vacuum by first heating the ligands from 0 K to 100 K over 50 ps. Ligand structures were simulated at 100 K for 100 ps. The simulation temperature was then allowed to drop back to 0 K over 50 ps. A 1-fs time step was used throughout the simulation. After simulated annealing, the ligand structures were energy-minimized with the MM+ force field until the energy gradient was less than 0.01 kcal/mol. Atomic partial charges for the _2A-AR_R models and small molecule ligands were assigned according to the Gasteiger method (19) implemented in Quanta 97 (Molecular Simulations Inc., San Diego, CA).

The program Autodock 2.4 was used to carry out docking simulations (20-22). The small molecule ligands were flexibly docked to the rigid frog rhodopsin-based receptor models. Autodock combines Monte-Carlo-simulated annealing for conformational searching with a rapid, atomic resolution, grid-based method of energy evaluation utilizing the AMBER forcefield (23, 24). The overall interaction between chemical species is estimated by using Lennard-Jones atom-atom potentials and electrostatic effects summed for the individual interactions between atoms. A distance-dependent dielectric constant was used to account for the solvent-screening effects. The interaction of a probe group (corresponding to each type of atom found in the ligand) with the wild type and mutant receptor models was computed at grid positions 0.35 Å apart in a 36-ų box centered at the binding site using Autogrid. In the second simulation step, a 20-ų box with grid positions 0.25 Å apart was used to refine the docked structures.

The parameters used in the simulation follow the "short schedule" described by Goodsell et al. (25). To make the simulation more thorough, the following changes were made to the short schedule. 1) 100 separate docking simulations were performed for each ligand; 2) for each simulation there were 100 constant temperature cycles with 3000 steps accepted or rejected; 3) the temperature was reduced by a factor of 0.97 in each cycle; and 4) the maximal torsional rotation and translation steps used were 15° and 0.2 Å, respectively, and they were reduced by a factor of 0.97 in each cycle. In this way, over 30 million conformations were studied for each ligand-receptor complex.

After docking, cluster analysis of the docked structures was carried out. A 1-Å cut-off value of the root mean square deviation calculated over all atoms in the ligands was used to define a new cluster. Bonding between the receptor molecules and the small molecules was investigated 1) using the optimal docked poses (i.e. conformation and orientation) found after this second stage of simulation and by 2) visualization on a graphics station together with the GRID maps. The docked pose that best fit the GRID maps was chosen as a representative pose. Thus, the representative pose for all the docked ligands was finally chosen by combining the docking results from Autodock with the GRID maps.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Comparison of the Bacteriorhodopsin and Frog Rhodopsin-based Models

From the five _2A-AR_BR (bacteriorhodopsin based) and the five _2A-AR_R (rhodopsin template based) models built with MODELLER (15), a single representative model was chosen for each with the lowest value of the MODELLER objective function: the model with the best agreement with all of the data used in the modeling process.

To evaluate the differences in the two predicted model structures, we first optimally superimposed the structures on each other and then evaluated how different the helix tilt angles were. The _2A-AR_BR and _2A-AR_R models were superimposed using the program VERTAA,² which revealed significant differences between the two models (Fig. 1, top). When all TM residues in the models were used in the comparison, 113 residues were superimposed with a root mean square deviation of 2.01 Å within the 3-Å cut-off, but none of the residues in TM4 were superimposed within this cut-off, because the closest distance between the equivalent C atoms in TM4 from the _2A-AR_BR and _2A-AR_R models is 3.8 Å. In the _2A-AR_R model, TM4 is more distant from TM3 and TM5 than in the _2A-AR_BR model (Fig. 1, top). This enables TM3 and TM5 to come closer to each other in the _2A-AR_R model than in the _2A-AR_BR model. TM4 of the _2A-AR_BR model tilts into the ligand binding cavity, and the helix tips in the _2A-AR_BR and the _2A-AR_R models are 3.8 Å apart at the extracellular side and 8 Å apart at the intracellular side (Fig. 1, top). The difference between the TM4 helix tilting angles in the _2A-AR_BR and the _2A-AR_R models is 10.8°.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Stereo views of the superimposition of the 2A-ARBR (red) and the 2A-ARR (blue) models. Top panels, top view from the extracellular surface of the receptor with the helices as cylinders. The 2A-ARR model is rotated clockwise compared with the 2A-ARBR model. Bottom panels, side view of the ligand binding site. Distances between the C atoms of Cys-201 and Asp-113 in the both models as well as the distance between Cys-201 in the 2A-ARBR and Cys-201 in the 2A-ARR models are indicated. All figures were prepared using the program MOLSCRIPT (28) and rendered using Raster3D (16).

The differences in the relative positions of the transmembrane helices in the _2A-AR_BR and _2A-AR_R models are larger at the ends of the helices than in the middle of them, and the helix tilt angles observed in the two models also differ. The differences between the helix tilt angles of TM3s, TM6s, and TM7s are smallest (5.9°, 5.8°, and 3.4°, respectively). The difference between the helix tilt angles is 9.9° for both TM1s and TM2s, and the difference is largest, 12.9°, between the tilt angles of TM5s. Generally, the differences in the tilt angles of the transmembrane helices in the two models can be described as a clockwise rotation of the _2A-AR_R model relative to the _2A-AR_BR model (Fig. 1, top).

As a result of the clockwise rotation of the helices of _2A-AR_R in comparison to _2A-AR_BR in the predicted structures, critical differences are seen in the relative positions of Asp-113 (TM3) and Cys-201 (TM5), the two key residues (10, 26, 27) implicated in ligand binding (Fig. 1B). The reactive aziridinium ion derivative of CEC forms a covalent bond with the sufhydryl side chain of Cys-201 and the carboxyl group of Asp-113 is, based on docking simulations, hydrogen bonding with the protonated imidazoline ring of CEC (Fig. 2, top and bottom) (9). In both the _2A-AR_BR and _2A-AR_R models, Asp-113 is located at approximately the same position in the superimposed model structures, whereas Cys-201 is much less exposed to the binding cavity in the _2A-AR_BR model than in the _2A-AR_R model (Fig. 1, bottom). The C atoms of Asp-113 and Cys-201 in the _2A-AR_R model, in comparison with _2A-AR_BR, are closer to each other, and they are positioned at about the same distance from the membrane boundaries (Fig. 1, bottom). Because of these differences in the helix positions and tilt angles of the two models, the ligand binding site at the extracellular end of the transmembrane helices is more exposed in the _2A-AR_R model than in the _2A-AR_BR model.

View larger version (59K): [in this window] [in a new window]   Fig. 2.   Stereo view of CEC and MTSEA binding to the 2A-ARBR model (top panels) and to the 2A-ARR model (bottom panels). The MTSEA·2A-ARBR complex is shown in red, and the MTSEA·2A-ARR complex is shown in blue. Both of the CEC·2A-ARBR complexes are shown in gray.

Because large differences exist between these two models, we can probe the ligand binding site using both computational methods and experimentally using the recombinant receptor and engineered mutant receptors. Thus, comparisons made between the two approaches can provide evidence in support of one model over the other. Alternatively, it is possible that neither model adequately reflects the ligand binding environment within _2A-AR, which also should be detectable.

CEC and MTSEA

Ligand Binding-- The second order rate constants for receptor alkylation with CEC and MTSEA were determined to quantitate the susceptibility of the engineered cysteines to these compounds. The consecutive amino acids extending from Val-197 to Ser-204 in TM5 were mutated to introduce or delete cysteines to examine the structure of TM5; the mutant receptors are designated _2A-ARSer201, _2A-ARSer201Cys197, _2A-ARSer201Cys200, _2A-ARSer201Cys202, _2A-ARSer201Cys203, and _2A-ARSer201Cys204. With the WT and the mutant receptors, _2A-ARSer201, _2A-ARSer201Cys197, and _2A-ARSer201Cys204, there was no significant difference in the alkylation rates obtained for CEC and MTSEA. The relative alkylation rate of the _2A-ARSer201Cys200 mutant, however, was much faster with CEC than, with MTSEA indicating that they may interact with different receptor conformations. The results obtained from reactions of the wild type and mutant receptors with CEC and MTSEA are described in complete detail elsewhere (9).

Docking Simulations-- To simulate the binding of ligand to the two predicted models, we manually docked CEC and MTSEA to the binding cavity of _2A-AR_BRWT, _2A-AR_RWT and to the mutant receptors derived from these model structures. In the wild type and mutant models based on bacteriorhodopsin, there are no differences seen in the accessibility of the reactive cysteine in TM5. Thus, Cys-200 in the _2A-AR_BRSer201Cys200 model is predicted to bind equally well to both CEC and MTSEA (Fig. 2, top).

In _2A-AR_RSer201Cys200, the accessibility of Cys200 to the ligands is, however, limited. Nonetheless, when the _2A-AR_RSer201Cys200·CEC complex is energy-minimized without constraints, TM5 was observed to adjust its orientation to allow proper ligand binding. As a result of this movement, covalent bonds can form between Cys-200 (TM5) and CEC, as well as hydrogen bonds between Asp-113 (TM3) and CEC (Fig. 2, bottom). In contrast, MTSEA is too short to form contacts with Asp-113 in TM3. Thus, when the _2A-AR_RSer201Cys200·MTSEA complex was energy-minimized without constraints, the orientation of TM5 did not change, and the disulfide bond length between Cys-200 and MTSEA was longer than the disulfide bond in the MTSEA complex with _2A-AR_RWT or with any of the other mutant receptors (Fig. 2, bottom).

Unlike the models based on the bacteriorhodopsin structure, the predicted results obtained for the models based on the frog rhodopsin template are in agreement with the experimental ligand binding studies for CEC and MTSEA. Because of these results, we have used the _2A-AR_R model, but not the _2A-AR_BR model, in all other docking studies reported here.

Other Ligands

Ligand Binding-- The results from the competition of [³H]RX821002 binding to the wild type _2A-AR and mutant receptors using clonidine, para-aminoclonidine, oxymetazoline, UK14,304, and norepinephrine as ligands are presented in Table II. K_iH and K_iL are inhibition constants for the high and low affinity sites in a statistically significant (p < 0.05) two-site model. The apparent K_i is the inhibition constant for a one-site model. Results for the _2A-ARSer201 mutant are presented only as apparent K_i values from one-site models as two-site fits were not statistically significantly better than them.

View this table: [in this window] [in a new window]   Table II Competition of [3H]RX821002 binding to 2AWT and mutant receptors, separately expressed in CHO cells KiH and KiL are inhibition constants for the high and low affinity sites in a statistically significant (P < 0.05) two-site model. Results for 2ASer201 mutant are statistically significantly modeled only with one-site fit. The Ki values are means ± S.E. from multicurve analysis of three separate experiments performed in triplicate. n.s., not significant.

Docking Simulations-- Five ligands, clonidine, para-aminoclonidine, oxymetazoline, UK14,304, and norepinephrine, which do not form covalent complexes in their binding to _2A-AR, were automatically docked to each of the wild type and mutant models. Thus, we have combined the docking results from Autodock with the GRID maps to choose the representative pose for each of these ligands. In _2A-AR_RWT, the optimal docked poses of all five ligands are similar (Fig. 3). The CH₂ groups in the imidazoline ring of clonidine, para-aminoclonidine, UK14,304, and oxymetazoline pack against TM7 (Fig. 3, top and middle). The imidazoline ring of clonidine, para-aminoclonidine, oxymetazoline, and UK14,304 is placed so that the ligands can form ideal hydrogen bonds with Asp-113. In oxymetazoline, the NH group of the imidazoline ring can make one bifurcated hydrogen bond with both side-chain oxygens of Asp-113 (Fig. 3, middle). Clonidine, para-aminoclonidine, and UK14,304 can make bidentate hydrogen bonds with the side-chain oxygens of Asp-113: one with the NH group in the imidazoline ring and the other one with the NH group in the aliphatic chain (Fig. 3, top). As a result, the imidazoline ring in clonidine, para-aminoclonidine, and UK14,304 is pushed slightly toward TM7 (Fig. 3, top). GRID maps also indicate that the TM7 region is favorable for hydrophobic CH₂ and CH₃ contacts.

View larger version (56K): [in this window] [in a new window]   Fig. 3.   Stereo view of UK14,304, para-aminoclonidine and clonidine (top panels) oxymetazoline (middle panels), and norepinephrine (bottom panels) docked to the 2A-ARRWT model.

In norepinephrine, there is an OH group present instead of the aliphatic NH group seen in the other ligands, but it is similarly positioned. As a result, the OH group can also make a favorable hydrogen bond with a side chain oxygen of Asp-113 (Fig. 3, bottom). With an OH group probe, the GRID maps calculated for the _2A-AR_RWT, _2A-AR_RSer201, and _2A-AR_RSer201Cys197 models indicate favorable interactions with the side-chain oxygens of Asp-113.

The aromatic ring, present in all of the docked ligands, is placed between TM3 and TM6, approximately parallel to TM3. GRID calculations predict favorable aromatic -CH group interactions at this site. In clonidine and para-aminoclonidine, there are two Cl atoms present in each; one pointing toward the extracellular membrane surface (up in Fig. 3, top) and the other one pointing downward in the binding cavity (down in Fig. 3, top). In UK14,304, a Br atom is present and preferentially points up even though it could be oriented either up or down. The methyl groups of oxymetazoline are pointing in a similar way as the Cl atoms in clonidine and para-aminoclonidine. The GRID calculations made using the CH₃ and hydrophobic probes indicate that hydrophobic contacts could be formed between the bottom of the binding cavity and the methyl group pointing down in oxymetazoline (Fig. 3, middle). The largest hydrophobic zone calculated with GRID is located near Cys-201 in TM5. Thus, the longer ligands, oxymetazoline and UK14,304, can form stronger hydrophobic contacts with Cys-201 of _2A-AR_RWT than the shorter ligands can (norepinephrine, para-aminoclonidine, and clonidine).

The cysteine residue in TM5 of the wild type receptor model seems to be important for ligand binding, since all of the docked ligands are always oriented toward that cysteine. In _2A-AR_RSer201, where cysteine contacts do not exist, the docked conformation of UK14,304 enforces the hydrogen bonding with Asp-113 (Fig. 4). This is true for the docking simulations of the other ligands too. Based on the GRID calculations, the perturbation caused by the replacement of Cys-201 with Ser makes the binding site environment slightly less hydrophobic. In _2A-AR_RSer201Cys197, the docked conformation of UK14,304 is distinctly closer to the membrane boundary than in the WT and other mutant receptors, and contacts are formed with both ends of the ligand (Fig. 4). In _2A-AR_RSer201Cys197, the distance from Asp-113 to Cys-197 is about 3.5 Å less than the distance from Asp-113 to Cys-201 in _2A-AR_RWT. Thus, in _2A-AR_RSer201Cys197 both ends of para-aminoclonidine and clonidine can form contacts with the receptor. Our docking simulations suggest that UK14,304 binds closer to TM5 in _2A-AR_RSer201Cys200 than in _2A-AR_RSer201, because the ligand can then form contacts with Cys200 in _2A-AR_RSer201Cys200 (Fig. 4). In _2A-AR_RSer201Cys200, Cys200 is pointing slightly away from the binding cavity, and thus UK14,304 can form only one hydrogen bond with Asp-113 (Fig. 4). In _2A-AR_RSer201Cys204, the cysteine is one turn lower but pointing in the same direction in the binding cavity as Cys-201 in the wild type receptor. Thus, UK14,304 (Fig. 4) and the other ligands can bind to _2A-AR_RSer201Cys204 in a similar way as seen in the wild type receptor.

View larger version (59K): [in this window] [in a new window]   Fig. 4.   UK14,304 docked sequentially to the wild type (Cys-201 in TM5), 2A-ARRSer201, 2A-ARRSer201Cys197, 2A-ARRSer201Cys200, and 2A-ARRSer201Cys204 mutant receptors.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have compared the bacteriorhodopsin (_2A-AR_BR)- and the rhodopsin-based (_2A-AR_R) models of human _2A-AR and combined the comparison with the CEC- and MTSEA-induced receptor alkylation studies using both the wild type (Cys-201 in TM5) and the mutant receptors having engineered cysteines in TM5. In the alkylation reaction, the reactive aziridinium ion derivative of CEC forms a covalent bond, and MTSEA forms a disulfide bond, with the cysteine residue accessible in the binding cavity.

Generally, the difference between the two models can be described as a clockwise rotation of the _2A-AR_R model relative to the _2A-AR_BR model (Fig. 1, top). This results in a tighter and more surface-exposed ligand binding site in the _2A-AR_R model (Fig. 1, bottom). The results with the _2A-ARSer201Cys200 mutant further supported the rhodopsin-based model. We could not explain with the _2A-AR_BR model why the alkylation rate of _2A-ARSer201Cys200 was higher with CEC than with MTSEA, because in both the _2A-AR_BR·CEC and _2A-AR_BR·MTSEA complexes, Cys-200 remained equally accessible to the alkylating reagent. However, the difference can be explained with the _2A-AR_R model, where Cys-200 becomes more accessible because of the conformational change in the receptor, which allows CEC to have, simultaneously, a bidentate hydrogen bond to Asp-113 and a covalent bond to Cys-200. MTSEA is covalently bonded to Cys-200 and is too short to reach the vicinity of Asp-113, and thus, it does not cause any conformational change in the receptor (Fig. 2). Based on these results, we have used the _2A-AR_R models in docking studies involving five other known ligands of _2A-AR but not the bacteriorhodopsin-based models.

We have carried out extensive ligand binding simulations on _2A-AR_RWT, _2A-AR_RSer201, _2A-AR_RSer201Cys197, _2A-AR_RSer201Cys200, and _2A-AR_RSer201Cys204 and compared the results with the experimental binding data. Cys-201 and Asp-113 have both been shown experimentally to be important for ligand binding in _2A-AR (26, 27). Our theoretical docking studies of clonidine, para-aminoclonidine, oxymetazoline, UK14,304, and norepinephrine also indicate the importance of these residues.

In _2A-AR_RWT, UK14,304, and oxymetazoline form interactions with both Asp-113 and Cys-201, whereas norepinephrine, clonidine, and para-aminoclonidine mainly contact Asp-113. In _2A-AR_RSer201Cys197, the distance between Asp-113 and Cys-197 is shorter than in the wild type receptor, and therefore, all of the ligands form strong interactions with the receptor, but the docked conformations are oriented in a very different way than in the other receptor models. Because the distance from Asp-113 to the cysteine residue in TM5 is longer in the _2A-AR_RSer201Cys200 and _2A-AR_RSer201Cys204 receptor models, the ligands are slightly too short to form interactions as strong as those seen in the wild type receptor. However, even though the distance is longer between Asp-113 and the Cys residue in TM5, the ligand interacts with the cysteine residue, not the serine, in TM5. Indeed, the docked ligands clearly prefer to interact with the cysteine residue in TM5 in each of the mutant receptors via the aromatic rings or other hydrophobic groups of the ligands. Furthermore, this is supported by the experimental binding results (Table II), which show a decrease in the binding affinity of all the ligands to _2A-AR_RSer201.

The binding affinity of UK14,304 to _2A-AR_RSer201 is nearly 200 times lower than to the wild type receptor (Table II). The favorable hydrophobic interactions between the hydrogen atoms of the aromatic ring in UK14,304 and the cysteine residue (Cys-201) have disappeared, a likely cause of this decrease in the binding affinity. In the _2A-AR_RSer201Cys197 and _2A-AR_RSer201Cys200 mutant receptors, the bidentate hydrogen bond between the ligand and Asp-113 is broken to allow the ligand to interact with both the cysteine residue in TM5 and Asp-113 in TM3 (Fig. 4). In addition, the orientation of UK14,304 changes and a hydrophobic interaction is formed between the aromatic ring of UK14,304 and the cysteine residue (Fig. 4). This could explain the lower binding affinities observed for these two mutant receptors for UK14,304 (Table II). In _2A-AR_RSer201Cys204, the docked conformation of UK14,304 is close to the conformation seen in the wild type receptor, but the bidentate hydrogen bond to Asp-113 is broken (Fig. 4). As a result, the decrease in binding affinity is smaller compared with the two other mutant receptors (Table II).

Norepinephrine similarly forms a bidendate hydrogen bond with Asp-113 as was predicted for UK14,304. However, the bidentate interaction is formed between the oxygen atoms of Asp-113 and the hydrogen atom of the NH₂ group and the -hydroxyl of norepinephrine (Fig. 3, bottom). Norepinephrine is smaller in size than UK14,304, and thus, norepinephrine does not ideally interact with the cysteine residues in the _2A-AR_RSer201Cys204 and _2A-AR_RSer201Cys200 mutant receptors. The binding affinities to these mutant receptors are lower than to the wild type receptor (Table II). In _2A-AR_RSer201Cys197, the cysteine residue interacts with the phenyl ring of norepinephrine, but the bidentate hydrogen bond with Asp-113 is lost, and therefore, the binding affinity is lower than observed with the wild type but higher than seen with the other two mutant receptors. Because the phenolic hydroxyl group of norepinephrine interacts with Ser-201, the difference in the binding affinity of norepinephrine with _2A-AR_RSer201 and with the wild type receptor is smaller than the corresponding difference for UK14,304 (Table II).

Oxymetazoline does not have the two NH groups needed to form a bidentate hydrogen bond with Asp-113. Instead, the NH group of the imidazoline ring forms hydrogen bonds with both oxygen atoms of Asp-113 (Fig. 3, middle). Oxymetazoline is larger than the other ligands, having a tert-butyl group attached to the phenyl ring, which makes favorable hydrophobic interactions with the cysteine residue in TM5. In _2A-AR_RSer201, the decrease in binding affinity is not as large as with UK14,304, because oxymetazoline, like norepinephrine, has a hydroxyl group that interacts with Ser-201. In _2A-AR_RSer201Cys197, the cysteine residue strongly interacts with the aromatic ring of oxymetazoline, and the hydrogen bond with Asp-113 is lost. Consistent with these modeling results, the observed binding affinity of oxymetazoline to _2A-AR_RSer201Cys197 is lower than to the wild type receptor (Table II).

Clonidine and para-aminoclonidine do form a bidentate hydrogen bond with Asp-113 in the wild type receptor model, but they are too short to form favorable interactions with the cysteine residue in TM5 too (Fig. 3, top). Similarly, they do not favorably interact with Ser-201 in _2A-AR_RSer201, and the observed change in the binding affinity compared with the wild type receptor is not large (Table II). However, in _2A-AR_RSer201Cys197, the distance between Asp-113 and Cys-197 is shorter than in the wild type receptor, and this permits simultaneous interactions between ligands and these important residues. However, only a single hydrogen bond between the NH group of the imidazoline ring and Asp-113 is formed instead of the bidentate hydrogen bond existing in the wild type receptor complexes. In addition, a hydrophobic interaction between the aromatic ring of both ligands and Cys-197 is formed. Consistent with these modeling results, only a slight increase in binding affinity can be seen (Table II).

In this work, we have used two programs: Autodock 2.4 (20-22) to simulate the docking of ligands to the receptor models, and GRID Version 16 (18), to probe chemically favorable interaction sites in the ligand binding site in the receptor models, and then combined these results to construct the ligand-protein complexes. Even though the binding site in the receptor is rigid in our docking simulations, the use of GRID maps allows us to examine the flexibility of the receptor binding site. In this way, the effects induced by the mutated residue in the vicinity of the binding site could be revealed, and the experimentally observed changes in binding affinity were explained at the atomic level. The combined use of molecular modeling methods and experimental data provides us with a more detailed understanding of ligand binding in the _2A-adrenergic receptor.

	ACKNOWLEDGEMENTS

We thank Professor Joyce Baldwin (Medical Research Council (MRC) Laboratory of Molecular Biology, MRC Center, Cambridge, UK) for kindly providing us the C atom template of the transmembrane helices of the rhodopsin-like GPCRs.

	FOOTNOTES

^* This study was supported by grants from the Academy of Finland, from the Technology Development Center in Finland (TEKES), and from the Erna and Victor Hasselblad Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Co-first authors of this paper.

^¶ Currently Postdoctoral Fellows of the Academy of Finland.

To whom correspondence should be addressed: Dept. of Biochemistry and Pharmacy, Åbo Akademi University, Tykistökatu 6A, FIN-20520 Turku, Finland. Tel.: +358 2 2154006; Fax +358 2 2154745; E-mail: tiina.salminen@btk.utu.fi.

² J. Lehtonen and M. S. Johnson, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: _2A-AR, human _2A-adrenengic receptor; _2A-AR_BR, bacteriorhodopsin-based models of human _2A-AR; _2A-AR_R, rhodopsin-based model of human _2A-AR; CEC, chloroethylclonidine; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; UK14,304, 5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; GPCR, G-protein-coupled receptor; TM, transmembrane helix/helices; WT, wild type; [³H]RX821002, [³H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1.	Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Backmann, E., and Downing, K. H. (1990) J. Mol. Biol. 213, 899-929[Medline] [Order article via Infotrieve]
2.	Schertler, G. F. X., Villa, C., and Henderson, R. (1993) Nature 362, 770-772[CrossRef][Medline] [Order article via Infotrieve]
3.	Pebay-Peyroula, E., Rummel, G., Rosenbusch, J. P., and Landau, E. (1997) Science 277, 1676-1681[Abstract/Free Full Text]
4.	Unger, V. M., Hargrave, P. A., Baldwin, J. M., and Schertler, G. F. X. (1997) Nature 389, 203-206[CrossRef][Medline] [Order article via Infotrieve]
5.	Baldwin, J. M., Schertler, G. F. X., and Unger, V. M. (1997) J. Mol. Biol. 272, 144-164[CrossRef][Medline] [Order article via Infotrieve]
6.	Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G., and Cotecchia, S. (1996) EMBO J. 15, 3566-3578[Medline] [Order article via Infotrieve]
7.	Schwartz, T. W. (1994) Curr. Opin. Biotechnol. 5, 434-444[CrossRef][Medline] [Order article via Infotrieve]
8.	Samana, P., Cotecchia, S., Costa, T., and Lefkowitz, R. J. (1993) J. Biol. Chem. 268, 4625-4636[Abstract/Free Full Text]
9.	Marjamäki, A., Frang, H., Pihlavisto, M., Hoffrén, A.-M., Salminen, T., Johnson, M. S., Kallio, J., Javitch, A., and Scheinin, M. (1999) J. Biol. Chem. 274, 21867-21872[Abstract/Free Full Text]
10.	Marjamäki, A., Pihlavisto, M., Cockcroft, V., Heinonen, P., Savola, J.-M., and Scheinin, M. (1998) Mol. Pharmacol. 53, 370-376[Abstract/Free Full Text]
11.	Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Feng, T. L., Francke, U., Caron, M. G., Lefkowitz, R. J., and Regan, J. W. (1987) Science 238, 650-656[Abstract/Free Full Text]
12.	Javitch, J. A., Fu, D., and Chen, J. (1995) Biochemistry 34, 16433-16439[CrossRef][Medline] [Order article via Infotrieve]
13.	Halme, M., Sjöholm, B., Savola, J.-M., and Scheinin, M. (1995) Biochim. Biophys. Acta 1266, 207-214[Medline] [Order article via Infotrieve]
14.	Fraser, C. M., Arakawa, S., McCombie, W. R., and Venter, J. C. (1989) J. Biol. Chem. 264, 11754-11761[Abstract/Free Full Text]
15.	ali, A., and Blundell, T. L. (1993) J. Mol. Biol. 234, 779-815[CrossRef][Medline] [Order article via Infotrieve]
16.	Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524[Medline] [Order article via Infotrieve]
17.	Gerstein, M. (1992) Acta Crystallogr. Sec. A 48, 271-276[CrossRef]
18.	Goodford, P. J. (1985) J. Med. Chem. 28, 849-857[CrossRef][Medline] [Order article via Infotrieve]
19.	Gasteiger, J., and Marsili, M. (1980) Tetrahedron 36, 3219-3228[CrossRef]
20.	Goodsell, D. S., and Olson, A. J. (1990) Proteins Struct. Funct. Genet. 8, 195-202 [CrossRef][Medline] [Order article via Infotrieve]
21.	Morris, G. M., Goodsell, D. S., Huey, R., and Olson, A. J. (1996) J. Comput. Aided Mol. Des. 10, 293-304[CrossRef][Medline] [Order article via Infotrieve]
22.	Goodsell, D. S., Morris, G. M., and Olson, A. J. (1996) J. Mol. Recognit. 9, 1-5[CrossRef][Medline] [Order article via Infotrieve]
23.	Weiner, S. J., Kollman, P. A., and Case, D. A. (1986) J. Comput. Chem. 7, 230-252 [CrossRef]
24.	Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, K. M., Ferguson, D. M., Spellmeyer, D. C., Fox, T., Caldwell, J. W., and Kollman, P. A. (1995) J. Am. Chem. Soc. 117, 5179-5197[CrossRef]; Correction (1996) J. Am. Chem. Soc. 118, 2309
25.	Goodsell, D. S., Lauble, H., Stout, C. D., and Olson, A. J. (1993) Proteins Struct. Funct. Genet. 17, 1-10 [CrossRef][Medline] [Order article via Infotrieve]
26.	Link, R., Daunt, D., Barsh, G., Chruscinski, A., and Kobilka, B. (1992) Mol. Pharmacol. 42, 16-27[Abstract]
27.	Wang, C. D., Buck, M. A., and Fraser, C. M. (1991) Mol. Pharmacol. 40, 168-179[Abstract]
28.	Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950 [CrossRef]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				P. J. Pauwels, I. Rauly, and T. Wurch Dissimilar Pharmacological Responses by a New Series of Imidazoline Derivatives at Precoupled and Ligand-Activated {alpha}2A-Adrenoceptor States: Evidence for Effector Pathway-Dependent Differential Antagonism J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 1015 - 1023. [Abstract] [Full Text] [PDF]

				T. Wurch, J. Okuda, and P. J. Pauwels Reciprocal Modulation of alpha 2A-Adrenoceptor and Galpha o Protein States as Determined by Carboxy-Terminal Mutagenesis of a Galpha o Protein Mol. Pharmacol., October 1, 2001; 60(4): 666 - 673. [Abstract] [Full Text] [PDF]

				T. Nyrönen, M. Pihlavisto, J. M. Peltonen, A.-M. Hoffrén, M. Varis, T. Salminen, S. Wurster, A. Marjamäki, L. Kanerva, E. Katainen, et al. Molecular Mechanism for Agonist-Promoted alpha 2A-Adrenoceptor Activation by Norepinephrine and Epinephrine Mol. Pharmacol., April 16, 2001; 59(5): 1343 - 1354. [Abstract] [Full Text]

				G. J. Molderings, H. Bonisch, M. Bruss, J. Likungu, and M. Gothert Species-Specific Pharmacological Properties of Human {alpha}2A-Adrenoceptors Hypertension, September 1, 2000; 36(3): 405 - 410. [Abstract] [Full Text] [PDF]

M. S. Singer Analysis of the Molecular Basis for Octanal Interactions in the Expressed Rat I7 Olfactory Receptor Chem Senses, April 1, 2000; 25(2): 155 - 165. [Abstract] [Full Text]