Stabilization of the Human β2-Adrenergic Receptor TM4–TM3–TM5 Helix Interface by Mutagenesis of Glu1223.41, A Critical Residue in GPCR Structure

G protein-coupled receptor (GPCR) instability represents one of the most profound obstacles in the structural study of GPCRs that bind diffusible ligands. The introduction of targeted mutations at nonconserved residues that lie proximal to helix interfaces has the potential to enhance the fold stability of the receptor helix bundle while maintaining wild-type receptor function. To test this hypothesis, we studied the effect of amino acid substitutions at Glu1223.41 in the well-studied β2-adrenergic receptor (β2AR), which was predicted from sequence conservation to lie at a position equivalent to a tryptophan residue in rhodopsin at the 3,4,5 helix interface among transmembrane (TM) domains 3, 4, and 5. Replacement of Glu1223.41 with bulky hydrophobic residues, such as tryptophan, tyrosine, and phenylalanine, increases the yield of functionally folded β2AR by as much as 5-fold. Receptor stability in detergent solution was studied by isothermal denaturation, and it was found that the E122W and E122Y mutations enhanced the β2AR thermal half-life by 9.3- and 6.7-fold, respectively, at 37 °C. The β1AR was also stabilized by the introduction of tryptophan at Glu1473.41, and the effect on protein behavior was similar to the rescue of the unstable wild-type receptor by the antagonist propranolol. Molecular modeling of the E122W and E122Y mutants revealed that the tryptophan ring edge and tyrosine hydroxyl are positioned proximal to the helical break in TM5 introduced by the conserved Pro2115.50 and may stabilize the helix by interacting favorably with the unpaired carbonyl oxygen of Val2065.45. Conformational flexibility of TM5 is likely to be a general property of class A GPCRs; therefore, engineering of the TM4–TM3–TM5 interface at the 3.41 position may provide a general strategy for the stabilization of other receptors.