Interestingly, a different "switch" one helical turn below W6.48 was observed in crystal structures of the β2 adrenergic receptor. The experiments described in this chapter test the rotamer switch and the F6.44/I3.40 switch in the dopamine D2 receptor and also test W6.48 in the muscarinic M2. Tolerance of this significant structural and electrostatic perturbation helps inform the position of the W6.48 side chain within GPCR binding sites.
In their study that proposed the rotamer switch model for GPCR activation, Javitch and co-workers supported their model by mutagenesis of the β2-adrenergic receptor.11 In their model (Figure 3.2), C6.47 adopts the trans rotamer in the inactive state, contacts W6.48, and switches to the gauche(-) rotamer in the active state. Indeed, the C6.47T mutant of the β2 receptor showed enhanced constitutive activity (basal signal in the absence of agonist) and a reduced EC50. These include antagonist-bound structures of the β2-adrenergic receptor and the D3-dopamine receptor.5,7,28 As such, the role of C6.47 in activation of the D2 receptor and other GPCRs remains unclear.
GPCR crystal structures thought to represent active conformations did not show any of the side chain transitions predicted by the rotamer switch model. If such a salt bridge could be designed and functionally tolerated, it would help inform the preferred orientation of the W6.48 side chain. We were particularly interested in investigating W6.48 in the M2 muscarinic acetylcholine receptor and in the D2 dopamine receptor.
In contrast, attempts to incorporate W6.48[5-NH2CH2Trp] into the D2 receptor under otherwise identical conditions showed no response. A salt bridge designed for W6.48 in the “active” conformation (Figure 3.8) suggested by the 5-NH2CH2Trp rotamer switch pairs with Y5.48E. In the speculative case that the D2 mutant W6.48[5-NH2CH2Trp] expresses but is nonfunctional, an interesting contrast with the M2 receptor emerges.
This could explain the surprisingly good tolerance of this side chain in the M2 receptor. This is in sharp contrast to the large losses of function recorded for both stereoisomers in the D2 dopamine receptor. Note that the different W6.48 conformations. specifically, different χ2 values) are seen in the D3 and M2 structures.
This effect could be partially responsible for the greater loss of function observed for (S)-β-MeTrp in M2. The F5.47L mutation in the M2 receptor, which reduces the size of the side chain, resulted in a major loss of function (Table 3.5). The fact that both methyls strongly couple to the 5.47 side chain suggests that the gauche(-) χ1 angle seen for W6.48 in the crystal structure may be real.
Steric clashes in this active conformation of the receptor are predicted to cause loss of function.
Conclusions
Overall, the lack of predictability suggests that either our model of the active receptor binding site is incorrect or that mutations intended simply to alter sterics have unintended consequences. At this point, it is difficult to know the true energetic consequence of the mutation in the actual receptor. Steric and electrostatic influences of the local protein environment certainly complicate the simple conformational analysis used for Thr/aThr or Ile/aIle, or the more sophisticated χ1,χ2 energy analysis used for the β-methyl analogs in this chapter.
Such an approach was used by Voth and co-workers to propose χ1, χ2 rearrangements of W6.48 in the A2A adenosine GPCR.15 If extended to unnatural amino acid analogues at W6.48 or other protein sites, this approach can improve the interpretation of experimental data to inform activation mechanisms.
Experimental
The THF was removed in vacuo and the aqueous layer was extracted with dichloromethane (3x), washed with brine, dried over MgSO 4 and concentrated. Trifluoroacetic acid was added (1 ml) and the reaction was stirred under argon for 1 hour to obtain the reaction solution. 6-Nitroveratryloxycarbonyl (NVOC) chloride (158 mg, 0.574 mmol, 3 eq) dissolved in dioxane (6 mL) was added and the reaction solution was stirred for 4 hours.
Aqueous 1M NaOH (0.5 mL) was added and the reaction mixture was stirred for 20 hours at room temperature. Additional 1M NaOH (1 mL) was added and the reaction mixture was heated at 40 °C for 1.5 h, after which the reaction was terminated by TLC. Triethylamine (24 µL, 0.17 mmol, 3 equiv) was added and the reaction mixture was stirred at room temperature for 6 h.
The organic layer was extracted with 5% aqueous NaHCO3 (5x) and the combined aqueous NaHCO3 layers were acidified with aqueous HCl to pH 1.5, then extracted with Et2O (5x) and the combined ether layers were washed with brine, dried over Na2SO4, and concentrated. Trimethylacetyl chloride (0.595 mL, 4.8 mmol, 1.2 eq) was added dropwise, upon which a yellow precipitate formed, and the reaction suspension was stirred at -10°C for 1 h. The reaction was quenched by the addition of 0.2 M aqueous HCl (40 mL, 2 eq), and the THF was removed in vacuo, ethyl acetate was added, and this organic layer was washed with 0.2 M aqueous HCl, then 1 M aqueous NaHCO 3 (2x ) , then brine, dried over Na2SO4 and concentrated.
Both flasks were cooled to -78 °C and the solution of compound 17 was cannulated into the KHMDS solution. The compound 17/KHMDS solution was transferred to this flask using a transfer cannula and the solution was stirred at -78 °C for 5 min. The reaction was quenched by the addition of glacial acetic acid (0.368 mL, 6.44 mmol, 4.8 equiv.) and the solution was warmed to room temperature and stirred at room temperature for 4 hours.
THF was removed in vacuo, Et2O (15 mL) was added and the layers were separated after mixing. H2O was added (0.1 mL, 5.6 mmol, 24 equiv.) and this solution was stirred at room temperature for 14 h, after which it was concentrated in vacuo. The resulting solution was lyophilized to give 15.2 mg of crude compound 21 , which was carried on to the next step without further purification.
This solution was cooled to 0 °C, 6-nitroveratryloxycarbonyl (NVOC) chloride (19.3 mg, 0.07 mmol, 1 equiv) was added and the reaction solution was stirred at room temperature for 3 h. Triethylamine (11.6 µL, 0.083 mmol, 3 equiv) was added and the reaction mixture was stirred at room temperature for 2 h and then concentrated in vacuo.
A conserved aromatic lock for the tryptophan rotameric switch in TM-VI of seven-transmembrane receptors. Mutation of a single TMVI residue, Phe282, in the β2-adrenergic receptor results in structurally different conformations of the activated receptor. Phe(303) in TMVI of the alpha(1B)-adrenergic receptor is a key residue that links TM helical movements to G-protein activation.
