A Conserved Aromatic Lock for the Tryptophan Rotameric Switch in TM-VI of Seven-transmembrane Receptors^*

Material

The ghrelin and substance P peptides were purchased from Bachem (Bubendorf, Switzerland). Ar231453 was kindly provided by Lisbeth Elster (7TM Pharma).

Molecular Biology

Receptor cDNAs were cloned into the eukaryotic expression vector pCMV-Tag(2B) (Stratagene, La Jolla, CA) for epitope tagging of the proteins with a FLAG tag. Mutations were constructed by PCR using the overlap extension method as previously described (22). The PCR products were digested with the appropriate restriction endonucleases (BamHI and EcoRI), purified, and cloned into the pCMV-Tag(2B) vector. All PCR experiments were performed using Pfu polymerase (Stratagene, La Jolla, CA) according to the instructions of the manufacturer. All mutations were verified by restriction endonuclease mapping and subsequent DNA sequence analysis using an ABI 310 automated sequencer. The CAM mutation of the β₂-adrenergic receptor carries the following substitutions in the C-terminal part of the third intracellular loop: L266S, K267R, H269K, and L272A.

Transfections and Tissue Culture

COS-7 cells were grown in Dulbecco's modified Eagle's medium 1885 supplemented with 10% fetal calf serum, 2 mm glutamine, and 0.01 mg/ml gentamicin. Cells were transfected using the calcium phosphate precipitation method with chloroquine addition as previously described (23). The amount of cDNA (20 μg/75 cm²) resulting in maximal basal signaling was used for the dose-response curves.

Phosphatidylinositol Turnover Assay

One day after transfection, COS-7 cells were incubated for 24 h with 5 μCi of [³H]myo-inositol (catalog number PT6–271, Amersham Biosciences) in 1 ml of medium supplemented with 10% fetal calf serum, 2 mm glutamine, and 0.01 mg/ml gentamicin per well. Cells were washed twice in 20 mm HEPES buffer, pH 7.4, supplemented with 140 mm NaCl, 5 mm KCl, 1 mm MgSO₄, 1 mm CaCl₂, 10 mm glucose, 0.05% (w/v) fetal bovine serum, and were incubated in 0.5 ml of buffer supplemented with 10 mm LiCl at 37 °C for 30 min. After stimulation with various concentrations of ligands at 37 °C, cells were extracted with 10 mm formic acid followed by incubation on ice for 30 min. The resulting supernatant was purified on Bio-Rad AG 1-X8 anion exchange resin to isolate the negatively charged inositol phosphates. After application of the cell extract to the column, the columns were washed twice with GPI buffer (60 mm sodium formate and 100 mm formic acid) to remove glycerophosphoinositol. Inositol phosphates were eluded by the addition of elution buffer (1 mm ammonium formate, 100 mm formic acid). Determinations were made in duplicates. The columns containing AG 1-X8 anion exchange resin were regenerated by the addition of 3 ml of regeneration buffer (3 m ammonium formate, 100 mm formic acid) and 5 ml of water.

cAMP Assay

One day after transfection, COS-7 cells (2.5 × 10⁵ cells/well) were incubated for 24 h with 2 μCi of [³H]adenine (catalog number TRK 311, Amersham Biosciences) in 1 ml of Dulbecco's 1885 medium supplemented with 10% fetal calf serum, 2 mm glutamine, and 0.01 mg/ml gentamicin. Cells were washed twice and incubated for 15 min at 37 °C in 1 ml of freshly prepared binding buffer supplemented with 1 mm isobutylmethylxanthine (catalog number I5879, Sigma) and with various concentrations of ligands or with 50 μm forskolin. After incubation, cells were placed on ice, medium was removed, and cells were lysed with 1 ml of 5% (w/v) trichloroacetic acid, supplemented with 0.1 mm cAMP and 0.1 mm ATP, for 30 min. The lysis mixtures were loaded onto a Dowex 50W-X4 resin (catalog number 142-1351, Bio-Rad) column (poly-prep columns, catalog number 731-1550, Bio-Rad), which was washed with 2 ml of water and placed on top of alumina columns (catalog number A9003, Sigma) and washed again with 10 ml of water. The columns were eluted with 6 ml of 0.1 m imidazole (catalog number I0125, Sigma) into 15 ml of Optiphase Highsafe scintillator (Wallac, Boston, MA). Columns were reused up to 10 times. Dowex columns were regenerated by adding 10 ml of 2 n HCl followed by 10 ml of water; the alumina columns were regenerated by adding 2 ml of 1 m imidazole, 10 ml of 0.1 m imidazole, and finally 5 ml of water. Determinations were made in duplicates or triplicates.

Competition Binding Assays

Transfected COS-7 cells were transferred to culture plates 1 day after transfection at a density of ∼5,000 cells per well, aiming at 5–8% binding of the radioactive ligand. Two days after transfection competition, binding experiments were performed for 3 h at 4 °C using ∼25 pm of ³⁵S-MK-677 (provided by Andrew Howard (Merck)). Binding assays were performed in 0.1 ml of a 50 mm Hepes buffer, pH 7.4, supplemented with 1 mm CaCl₂, 5 mm MgCl₂, 0.1% (w/v) bovine serum albumin, and 40 μg/ml bacitracin. Nonspecific binding was determined as the binding in the presence of 1 μm unlabeled ghrelin. Cells were washed twice in 0.1 ml of ice-cold buffer, 50 μl of lysis buffer/scintillation fluid (30% ethoxylated alkylphenol and 70% diisopropyl naphthalene isomers) was added, and the bound radioactivity was counted. Determinations were made in triplicate. Initial experiments showed that steady state binding was reached with the radioactive ligand under these conditions.

Cell Surface Expression Measurement (ELISA)

Cells were transfected and seeded out in parallel with those used for immunoprecipitation or cAMP accumulation assays. The cells were washed twice, fixed, and incubated in blocking solution (phosphate-buffered saline plus 0.2% dry milk) for 60 min at room temperature. Cells were kept at room temperature for all subsequent steps. Cells were incubated for 2 h with anti-FLAG (M2) antibody (Sigma) in a 1:300 dilution. After three washes, cells were incubated with anti-mouse horseradish peroxidase (Amersham Biosciences)-conjugated antibody at a 1:4000 dilution. After extensive washing the immunoreactivity was revealed by the addition of horseradish peroxidase substrate according to the manufacturer's instructions.

Calculations

IC₅₀ and EC₅₀ values were determined by nonlinear regression using the Prism 3.0 software (GraphPad Software, San Diego). The basal constitutive activity is expressed as a percentage of the ghrelin-induced activation for each mutant construct of the ghrelin receptor.

Molecular Dynamics Simulations

The methods used for both NAMD and Monte Carlo simulations are described only briefly here but in detail in the supplemental material.

NAMD Simulations

NAMD simulations were performed using either the 2.4 Å x-ray structure of B2AR (Protein Data Bank code 2RH1) (18, 24) or the 2.65 Å x-ray structure of bovine rhodopsin (Protein Data Bank code 1GZM) (25) as a starting structure where the carazolol and 11-cis-retinal ligands were removed. The receptors were manually inserted into a hexagonal membrane surrounded by water, and structural water molecules from the x-ray structures were included. All calculations were carried out with the NAMD simulation package (26) using the CHARMM27 force field (27). Simulations were generally performed for 8 or 20 ns.

Monte Carlo Simulations

Monte Carlo simulations were performed on the B2AR (Protein Data Bank code 2RH1) structure in a setup similar to that used for the NAMD simulation but with Phe-VI:17 rotated away from the binding pocket to its other preferred conformation to allow for free movement of Phe-V:13 and Trp-VI:13. The model of the active structure of B2AR was generated by a Monte Carlo simulated annealing protocol using distance constraints applied by the nuclear Overhauser effect functionality of CHARMM corresponding to Cβ-Cβ nuclear Overhauser effect distance constraints between positions III:08, VI:16, and VII:06 based on the activating metal ion site engineered into B2AR (28), as described for the CXCR3 receptor (29). The distance constraints for the intracellular segments of the transmembrane helices were based on the extensive double electron-electron resonance spectroscopy analysis of double spin-labeled rhodopsin (30) (see the supplemental material for details). Distance constraints were also applied between Phe-V:13 and Trp-VI:13 to ensure aromatic stacking of the two side chains (31). To allow for TM-V, TM-VI, and TM-VII to change helical kink conformation, ϕ, ψ backbone torsion angles of Trp-VI:13, which are responsible for the pronounced kink in TM-VI, were allowed to be perturbed to obtain energetically favorable conformations to stabilize the helix locally; similarly, the backbone ϕ, ψ torsion angles from Val-VII:11 to Phe-VII:15 were allowed to be perturbed in TM-VII, and the backbone ϕ, ψ torsion angles of Phe-V:13 were allowed to be perturbed in TM-V. The CHARMM program package and the CHARMM27 force field (27) were used to simulate the activation mechanism, and the Monte Carlo command (MOVE ADD) in CHARMM was used to construct the move sets (32). During the simulation protocol, perturbations were applied to translations, rotations, and torsional degrees of freedom for the defined move sets. The backbone of TM-I, -II, -III, and -IV were held fixed throughout the entire simulation. To allow for the TM-V, TM-VI, and TM-VII to move compared with each other, perturbation of the backbone ϕ, ψ angles of the ICL2 (residues 226–270) and ECL2 (residues 298–309) and the loop between TM-VII and helix VIII and helix VIII was allowed (residue 329–342). Perturbation of all side chains was allowed. Additional side-chain perturbation move sets were added for Trp-VI:13, Phe-V:13, and Phe-VI:17 in order for these residues to move more frequently than the other side chains in the receptor.

Molecular Dynamics Simulation of the Trp-VI:13 Rotamer Switch

Molecular dynamics simulations were performed with either rhodopsin (Protein Data Bank code 1GZM) or B2AR (Protein Data Bank code 2RH1) in a hexagonal simulation box with the receptors each placed in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane surrounded by water on both sides, as described under “Experimental Procedures.” It should be noted that this type and length (up to 20 ns) of molecular dynamics simulations are particularly suited to studying side-chain movements but not, for example, large scale helical movements. The inverse agonist ligands, 11-cis-retinal and carazolol, were in most instances removed to allow Trp-VI:13 to move as freely as possible.

In rhodopsin with the inverse agonist, 11-cis-retinal still present in the structure, Trp-VI:13 (as expected) did not change rotameric state, as illustrated by its very stable chi1 angle observed throughout 8 ns of molecular dynamics simulation (supplemental Fig. 1). However, the removal of 11-cis-retinal allowed Trp-VI:13 to change rotameric state during the molecular dynamics simulations with fluctuations of up to 80° in its chi1 angle from a starting position of −70° to approximately −150°, a rotameric state that was only visited occasionally (Fig. 2a). In the retinal-free rhodopsin structure, Phe-V:13 also probed various rotameric states during the molecular dynamics simulations. In the simulation shown in Fig. 2, the side chains of Trp-V:13 and Phe-V:13 established a relatively close interaction after ∼9 ns of molecular dynamics simulation, albeit with variable geometry (Fig. 2b). As shown in Fig. 2c, Trp-VI:13 and Phe-V:13 occasionally did form ideal aromatic stacking interactions, which only lasted for a short period in the retinal-free rhodopsin structure (Fig. 2a).

During similar molecular dynamics simulations of the B2AR, after removal of the inverse agonist carazolol, Trp-VI:13 was surprisingly stable, with fluctuations in its chi1 angle of only up to approximately ±15° around the starting position of −80°. This pattern was rather similar to the limited fluctuations in the chi1 angle of Trp-VI:13 observed in rhodopsin before removal of 11-cis-retinal (supplemental Fig. 1). The stability of the indole side chain of Trp-VI:13 in B2AR appeared to be related to its hydrogen bond interaction with a structural water molecule, which was stabilized by the side chains of Asp-II:10 and Ser-VII:13, of which the latter is not present in rhodopsin (supplemental Fig. 1b).

These molecular dynamics simulations indicate that upon removal of the inverse agonist ligand, Trp-VI:13 can (at least in rhodopsin) rotate into a conformation where its side chain is in rather close proximity to the equally highly conserved Phe-V:13. However, in the overall inactive state of the receptor, this interaction appears to be far from ideal and stable.

Probing the Proximity of Trp-VI:13 to Phe-V:13 by Metal Ion Site Engineering

At the most extracellular ends of TM-V and TM-VI, we have previously constructed inhibitory, interhelical metal ion sites between positions V:01, V:05, and VI:24 in, for example, the NK1, the κ-opioid, and the ghrelin receptors (33–35) (Fig. 1a). In the present study, we performed metal ion site engineering 1½ to 2 helical turns “deeper” into the membrane, where a His residue is found at position VI:17 in the wild-type ghrelin receptor (Fig. 1). Substitution of Phe-V:13 or Trp-VI:13 individually with His residues in each case had only a minor effect on the affinity of Zn²⁺ as determined in competition binding experiments against the radiolabeled non-peptide agonist ³⁵S-MK677 (Table 1 and Fig. 3b). This indicates that a His residue in position V:13 or VI:13 can form a metal ion site with His-VI:17 only to a limited extent. Importantly, however, combination of the two substitutions in the FV:13H/WVI:13H double mutant increased Zn²⁺ affinity to 36 ± 7 μm (Table 1 and Fig. 3c). This affinity is similar to previous observations for several bidentate metal ion sites in various receptors (33–35). This indicates that His-V:13 and His-VI:13 presumably can form a bidentate metal ion site with each other (Fig. 3c, right). The Zn²⁺ affinity was slightly further increased to 24 ± 7 μm by introducing a Cys residue at position V:13 (i.e. the FV:13C/WVI:13H double mutant) (Fig. 3d).

In none of the metal ion site engineered ghrelin receptor constructs did Zn²⁺ stimulate signal transduction as determined by inositol phosphate turnover (data not shown). Thus, the engineered metal ion binding sites between positions VI:13 and V:13 were not able to mimic the presumed aromatic-aromatic interaction between Trp-VI:13 and Phe-V:13 of the active receptor conformation. Consequently, it could be assumed that the high affinity Zn²⁺ binding instead would be associated with inverse agonism and/or antagonism. However, further functional analysis of the metal ion site engineered ghrelin receptor was hampered by the fact that the constitutive activity of the receptor was eliminated (see below) and that ghrelin could not activate signaling in these constructs (Table 2), despite the fact that both ghrelin and the non-peptide agonist MK677 bound with normal high affinity (Table 1 and Fig. 3).

These experiments support the notion that positions V:13 and VI:13 are in close spatial proximity (i.e. close enough to form high affinity metal ion sites when appropriately mutated with metal ion-binding residues).

Functional Analysis of Trp-VI:13 in the Ghrelin Receptor

Ala or His substitution of Trp-VI:13 in the ghrelin receptor did not affect the surface expression of the receptor in transfected COS-7 cells as judged by the ELISA directed against the N-terminally tagged receptor (Fig. 4a, inset). The WVI:13A and WVI:13H mutants also bound the agonist ghrelin with almost normal affinity, as shown in competition binding experiments against ³⁵S-MK677, K_i = 0.71 versus 0.75 nm for the wild-type receptor (Fig. 4a and Table 1). However, the high constitutive activity of the ghrelin receptor, which in respect to immunoprecipitation signaling normally is ∼45% of the maximal signaling capacity (35), was totally eliminated by the Ala substitution of Trp-VI:13 (Fig. 4b and Table 2).

The agonist-induced signaling efficacy of the WVI:13A mutant was also strongly impaired, with an E_max for ghrelin being 24% (i.e. ∼10% of that observed in the wild-type receptor). The low efficacy was observed despite the fact that the mutant receptor was expressed at the cell surface even better than the wild-type receptor, as observed both by ELISA (Fig. 4a, inset) and B_max calculation from competition binding experiments (Table 1). The EC₅₀ for ghrelin was increased by ∼10-fold from 0.34 nm in the wild-type receptor to 3.2 nm in the WVI:13A mutant. An imidazole could not substitute for the indole side chain of Trp-VI:13 because the molecular pharmacological phenotype of the WVI:13H mutant was rather similar to that of the WVI:13A mutant form (i.e. including a totally eliminated constitutive activity) (Table 2). It is concluded that Trp-VI:13 is essential for the constitutive signaling of the ghrelin receptor and that it is also important for the agonist-induced signaling but, importantly, not for agonist binding as such.

Functional Analysis of His-VI:17 in the Ghrelin Receptor

In its presumed active rotameric form, Trp-VI:13 could potentially interact with either Phe-V:13 or His-VI:17 (Fig. 1). In position VI:17 of 7TM receptors, one often finds either a His residue (29%), as in the ghrelin and many other peptide receptors, or a Phe residue (20%). Removal of the side chain of His-VI:17 by Ala substitution decreased the constitutive activity of the ghrelin receptor to 39% of that of the wild type (Fig. 5a, right). However, both the potency and the maximal efficacy of ghrelin was unaltered by the His-VI:17 → Ala substitution (Fig. 5a and Table 2). It is concluded that His-VI:17 to some extent could be involved in the activation process (i.e. in particular the ligand-independent, basal signaling activity) but that this is not a critical residue because it is not required for agonist activation of the receptor.

Functional Analysis of Phe-V:13 in the Ghrelin Receptor

Phe-V:13 in the ghrelin receptor was substituted with Ala, His, Tyr, or Cys, and all mutants were well expressed at the cell surface (Fig. 5c). As was observed with substitution of the Trp-VI:13 rotameric switch itself, Ala substitution of Phe-V:13 totally eliminated the constitutive signaling of the ghrelin receptor (Fig. 5a, left). In the FV:13A construct, ghrelin stimulated signaling with normal potency but with reduced E_max. Although Tyr is found at position V:13 in 10% of 7TM receptors, the phenol could not in the ghrelin receptor substitute for the phenyl side chain of Phe-V:13 because the molecular pharmacological phenotype of the Tyr-V:13 construct was similar to the Ala-V:13 construct, albeit with a measurable but low degree of constitutive signaling (Fig. 5b, middle). Similarly, the introduction of His or Cys at position V:13 eliminated or seriously decreased the constitutive signaling and reduced the E_max for ghrelin (Fig. 5b, right and left). The binding affinity of the endogenous peptide agonist ghrelin was not seriously affected by these substitutions at position V:13 (Table 1). It is concluded that Phe-V:13 is essential for the high constitutive activity of the ghrelin receptor and that it also is important for the agonist-induced signaling efficacy but not for agonist binding affinity or potency.

Mutational Analysis of Trp-VI:13 and Phe-V:13 in Other 7TM Receptors

GPR119 is a 7TM receptor, which displays almost 50% constitutive signaling activity (i.e. similar to the ghrelin receptor) but through the G_s and not the G_q pathway (Fig. 6a). As observed in the ghrelin receptor, Ala substitution of Trp-V:13 totally eliminated the constitutive activity of GPR119 (Figs. 4 and 6a). The expression level of the WVI:13A GPR119 construct was decreased to 37% of that observed in the wild-type receptor (Fig. 6a, left, inset); however, other GPR119 mutants with similar expression level display normal, high constitutive activity (data not shown). Ala substitution of Phe-V:13 in GPR119 decreased the constitutive signaling and impaired the agonist-induced E_max without affecting the potency of the agonist ligand (Fig. 6a, right).

In GPR39, which is a member of the ghrelin receptor family and also signals with high constitutive activity through the G_q pathway (36), Ala substitution of Trp-VI:13 seriously impaired the expression of the receptor, which accordingly did not show any constitutive or agonist-induced signaling activity (Fig. 6b, left). In contrast, Ala substitution at position V:13, which in the case of GPR39 is a Tyr residue, did not affect the expression of the receptor but was associated with a clear reduction in both constitutive and agonist-induced signaling (Fig. 6b, right).

In the transfected COS-7 cells of the present study, the ligand-independent signaling of the B2AR was below the detection limit. However, as shown in Fig. 6c, Ala substitution of the Trp-VI:13 rotameric switch basically eliminated the agonist-induced signaling of the B2AR, whereas Ala substitution of Phe-V:13 reduced the E_max for pindolol⁵ to ∼25% of that observed in the wild-type receptor without affecting the agonist potency. The 15% constitutive activity of the so-called CAM variant of the B2AR was eliminated by Ala substitution of both Trp-VI:13 and Phe-V:13 (data not shown).

In the NK1 receptor, which is devoid of any constitutive signaling activity, Ala substitution of Trp-VI:13 almost eliminated substance P-induced signaling, whereas Ala substitution of Phe-V:13 impaired the E_max but only by ∼40% (Fig. 6d). As in the other receptors, this impairment in signaling efficacy was not associated with impairment of agonist potency.

It is concluded that in all of the receptors addressed in the present study, Ala substitution of the Trp-VI:13 rotameric switch itself eliminates constitutive signaling and strongly impairs agonist induced signaling without affecting agonist potency, where this could be measured. In the case of the proposed aromatic lock for the Trp-VI:13 microswitch, Phe-V:13, Ala substitution also in all cases impaired both the constitutive and the agonist-induced receptor signaling, but not to the same degree as observed in the constructs where Trp-VI:13 itself was mutated, but again without affecting agonist potency.

Improved Trp-VI:13-Phe-V:13 Interaction Is Observed in a Presumed Active Receptor Conformation

As shown in Fig. 2, although Trp-VI:13 and Phe-V:13 could reach each other during the molecular dynamics simulations, they were not able to establish a strong aromatic interaction in the inactive state of rhodopsin. However, such an interaction would occur preferentially in the active global toggle switch conformation of the receptor. Accordingly, we used Monte Carlo simulations combined with experimentally derived distance constraints to generate a model of a presumed active receptor conformation based on the B2AR high resolution x-ray structure. As described previously for rhodopsin-based models of the B2AR and the CXCR3 receptor (28, 29), distance constraints corresponding to the Cβ-Cβ distances for the activating metal ion binding sites built between positions III:08, VI:16, and VII:06 (29, 37) were used as nuclear Overhauser effect distance constraints for the proposed active conformation to encourage inward movement of the extracellular segments of TM-VI and TM-VII. For the intracellular segments, the distance constraints were based on the distance changes observed between a series of double spin-labeled rhodopsin constructs, as observed in the inactive (dark) versus the active (light) state as determined by double electron-electron resonance spectroscopy (30) (see supplemental material for details). Initial simulations indicated that the close aromatic interaction between Phe-V:13 and Phe-VI:17 found in the B2AR crystal structures where antagonists interact directly with these residues had to be disrupted for Trp-VI:13 to rotate fully and for it to form favorable aromatic interactions with Phe-V:13. For the molecular simulations, we therefore used a starting configuration where Phe-VI:17 had been manually rotated to another preferred rotameric state pointing out toward the lipid bilayer.

During the first 350,000 Monte Carlo steps, the extracellular end of TM-VI gradually moved inward toward TM-III to fulfill the distance constraints of the activating metal ion site (Fig. 7b). However, already after 140 steps, Trp-VI:13 rotated from its “vertical” starting position with a chi1 of −73° toward a more horizontal orientation pointing toward TM-V with a chi1 of approximately −140° (Fig. 7d). From this conformation, the chi1 angle of Trp-VI:13 gradually approached −180°, corresponding to the t-conformation. Already, the first rotation of Trp-VI:13 brought its indole side chain close to Phe-V:13 (i.e. from 7–8 Å to ∼4 Å) (Fig. 7c), after which the distance gradually narrowed to ∼3 Å, corresponding to an ideal aromatic stacking distance (31). At this point, the two aromatic side chains formed a perfect aromatic face-to-face stacking interaction (Fig. 7a). Despite the fact that the distance constraints forced or encouraged the extracellular end of TM-VI to tilt into the main ligand-binding pocket, the calculated interaction energy between TM-VI and the rest of the receptor only increased marginally, and interestingly, the energy in fact passed over an energy barrier to reach a new minimum after around 450,000 Monte Carlo steps (Fig. 7e). It is concluded that in a proposed active global toggle switch conformation with the extracellular end of TM-VI tilted inward toward TM-III, the side chain of Phe-V:13 can form an ideal aromatic interaction with the side chain of Trp-VI:13 when this is rotated into its proposed active t-conformation.

Phe-V:13-Trp-VI:13 Interactions

Even in the inactive conformation of rhodopsin, where 11-cis-retinal was removed, the indole side chain of Trp-VI:13 can rotate around its dihedral chi1 to reach a position where it interacts with the phenyl side chain of Phe-V:13 (Fig. 2). However, the molecular dynamics simulations indicate that a stable interaction is not established in this overall inactive state of the receptor. Unfortunately, the supposedly active structure of opsin in complex with the G protein fragment cannot be used as a model for the active conformation of the extracellular part of 7TM receptors because it is missing the agonist ligand, all-trans-retinal, and because it still has the extracellular “protein plug” arranged in an inactive-like conformation (7, 8, 10, 38–40). NMR spectroscopy indicates that the movement of the extracellular end of TM-VI is linked with a displacement of extracellular loop 2 (i.e. a change in the conformation of the protein plug) (38). In order to generate a model of an active receptor conformation, we used Monte Carlo simulations, which are particularly suited to simulate more large scale molecular dynamic excursions (32). Distance constraints from activating metal ion sites between TM-III, TM-VI, and TM-VII were applied for the extracellular segments, and constraints from Hubbell and co-workers (30) double electron-electron resonance experiments were used for the intracellular parts of the transmembrane segments. In these Monte Carlo simulations, where the extracellular segment of TM-VI is tilted into the main ligand-binding pocket, Trp-VI:13 could in its supposedly active, trans conformation make an ideal aromatic-aromatic interaction with Phe-V:13 (Fig. 7). We suggest that such an interaction between the equally highly conserved Phe-V:13 and Trp-VI:13 is part of the structural constraint involved in stabilizing the global, active conformation in 7TM receptors. This notion is supported by the deleterious effect observed in a number of different receptors on the spontaneous, constitutive signaling upon Ala substitution of not only Trp-VI:13 (the rotamer switch itself) but also upon Ala substitution of Phe-V:13 (i.e. the proposed lock for the active conformation of Trp-VI:13) (Figs. 4 and 6). These mutations also impaired the agonist-induced maximally achieved efficacy, importantly, however, without affecting the potency of the agonist. This, indicates that mutation of Trp-VI:13 or Phe-V:13 in these receptors does not affect ligand binding but instead selectively impairs the activation mechanism as such.

In fact, even before x-ray structures of 7TM receptors were available, it was suggested by Gershengorn and co-workers (16) that Trp-VI:13 and Phe-V:13 could be part of a common aromatic cluster. Initially, they proposed that this interaction constrained the receptor in its inactive conformation because disruption of this aromatic cluster through substitutions increased the constitutive signaling of their original model receptor, TRH R1 (16). Subsequently, they found that Ala substitution of Trp-VI:13 had the opposite effect in the TRH-R2 receptor, where it instead eliminated the high constitutive activity of this receptor (41), which we also observe in the present study for a number of other 7TM receptors: the ghrelin receptor, GPR39, GPR119, and the CAM form of the B2AR (Fig. 6). Similarly, the high constitutive activity of the 5HT-4 receptor is eliminated by Ala substitution of Trp-VI:13 (15, 42). However, this is not the case for the CB1 receptor, in which the constitutive activity basically is unaffected by Ala substitution of Trp-VI:13 (43) and in the B2 bradykinin receptor, where Trp-VI:13 also only plays a subtle role in controlling the balance between the active and inactive state of the receptor (44). Nevertheless, it can still be concluded that in the many cases where Ala substitution of Trp-VI:13 impairs the constitutive activity of the receptor, Ala substitution of Phe-V:13 has a similar effect. This supports the notion that the conserved Phe-V:13 is an important part of the Trp-VI:13 rotameric switch function.

It was generally expected that TM-V would participate in the global 7TM activation mechanism (2, 4, 45–48), which recently was confirmed by the relatively large movement of the intracellular segment of TM-V observed in the x-ray structure of opsin in complex with the G-protein peptide (7, 8). Various biophysical studies, including fluorescence spectroscopy of the B2AR, suggest that relocation of the extracellular part of TM-V is required to facilitate binding of the agonist (45). Also, NMR studies on rhodopsin have demonstrated movements of TM-V due to steric interaction with the β-ionone ring and coupling with extracellular loop-2 displacement from the binding pocket (38, 39). The interaction of the Trp-VI:13 rotameric switch with Phe-V:13 demonstrated in the present study could very well constitute an important molecular communication between TM-VI and TM-V during the 7TM receptor activation process.

Other Interaction Partners for Trp-VI:13

Position III:12 (3.36) is found right across from Trp-VI:13 in the main ligand-binding pocket and could, depending on the side chain, consequently also influence the function of the Trp-VI:13 rotameric switch. Importantly, in contrast to position V:13, which has been conserved as either a Phe or a Tyr in 81% of 7TM receptors, no particular type of residue has been conserved at position III:12 (49) (Fig. 1). In the 5HT4 receptor, concerted rotation of the side chain of position III:12 and Trp-VI:13 has been proposed to occur to adopt the active conformation (15). Similarly, in the histamine H1 receptor, a Ser in position III:12 is important for the constitutive activity of this receptor and has been suggested to act in a similar manner (11). Although Ala substitution of Thr-III:12 impairs the constitutive activity of these receptors, the endogenous agonists (i.e. 5HT and histamine, respectively) are still able to activate the receptors with full efficacy (11, 15). The constitutive activity of the CB1 and the ghrelin receptors is also affected by removal of the side chain in position III:12 (i.e. Phe and Thr, respectively). But in these two receptors, the constitutive activity is, however, increased instead of decreased by Ala substitution (43, 50). In these cases, it could be speculated that the space-generating Ala substitution across from Trp-VI:13 in the main ligand-binding pocket could facilitate the rotation of the indole side chain into its presumed active trans conformation. Thus, it may be concluded that the residue found in position III:12 often influences the function of the Trp-VI:13 rotameric switch and receptor function but that it does so in a highly context-dependent manner.

From several of the x-ray structures, it appears that the indole side chain of Trp-VI:13 in its inactive, rotameric g+ conformation interacts with a structural water molecule, which is part of the hydrogen bond network between TM-I, TM-II, TM-VI, and TM-VII (9, 11, 18, 24, 51). Thus, polar residues in position VII:12, which is 79% conserved as either an Asn or a Ser residue, and in particular position VII:13, which is 79% conserved as a Ser or a Cys residue, are probably indirectly involved in stabilizing the inactive state of Trp-VI:13 (11).

Blocking the Trp-VI:13 Rotamer Switch?

In rhodopsin, the inverse agonist 11-cis-retinal bends around TM-VI, interacting closely with Trp-VI:13, and thereby blocks any movement of this residue and TM-VI as such (2, 47, 52). However, in the novel x-ray structures of the β₂-adrenerigic, β₁-adrenergic, and adenosine A2a receptors, the co-crystallized antagonists/inverse agonists do not appear to have close interaction with or directly block Trp-VI:13 movement (9, 18–20, 24). It has even been argued that the fact that these compounds are not full inverse agonists could be related to their poor prevention of the Trp-VI:13 rotamer switch function (6). Importantly, however, for example, carazolol, timolol, and cyanopindolol all interact closely with the aromatic “sandwich” formed by Phe-V:13 and Phe-VI:17 (18–20, 24). This aromatic cluster blocks Trp-VI:13 from rotating into its presumed active, trans conformation, as observed in the molecular dynamics simulations of the present study.⁶ Thus, although the antagonists/inverse agonists do not directly interact with the Trp-VI:13 rotameric switch, they apparently instead block its function indirectly by participating in an aromatic cluster between TM-V and TM-VI. Moreover, they all block the proposed inward movement of TM-VI, i.e. they block the proposed global toggle switch activation mechanism (9). It has also been proposed that partial agonism can be achieved by ligands without engaging the Trp-VI:13 rotameric switch, whereas full agonism appears to require this conformational change (6, 53).

30% of 7TM Receptors Function without Trp-VI:13

It may be somewhat surprising that Trp-VI:13, which often is considered to be an essential part of the activation mechanism for 7TM receptors, in fact only is found in 71% of these receptors (49). In 19% of the cases, position VI:13 is occupied by another aromatic residue (Phe or Tyr). However, around 10% of all 7TM receptors function with a non-aromatic residue, such as Gln, in position VI:13 (e.g. many chemokine receptors) (49).

Notably, the fact that a relatively large fraction of 7TM receptors function well without the Trp-VI:13 rotameric switch is only a problem in “sequential mode” allosteric activation models, where it would constitute a missing “domino brick” in the sequential line of intramolecular events connecting extracellular agonist binding with intracellular G protein activation (9, 11, 54). In contrast, in a Monod-Wyman-Changeux type of “concerted action” allosteric model, the Trp-VI:13 rotameric switch would just be one of several microswitches that form an extended allosteric interface between the transmembrane helices performing the global toggle switch movements that mediate the intramolecular signal transduction signal (9). In such a model, any of the microswitches are in principle dispensable, provided that the active conformation is still stabilized by other parts of the allosteric interface. Thus, during the gradual evolutionary process, different parts of the allosteric interface could in certain receptors have been strengthened, thereby allowing other parts of the interface to be weakened. However, in the majority of receptors where a Trp residue is found in position VI:13, an experimental, “acute” substitution of this has a dramatic effect on both spontaneous and agonist-induced activation, as does substitution of its aromatic lock, Phe-V:13. Variations in the setting of the different microswitches of the allosteric interface, such as the Trp-VI:13 rotameric switch and the water hydrogen bond network, may be highly important for fine tuning the signal or be involved in controlling, for example, signal transduction pathway-biased signaling (9).