Abstract

The binding free energy for the interaction between serines 204 and 207 of the fifth transmembrane helix of the β₂-adrenergic receptor (β₂-AR) and catecholic hydroxyl (OH) groups of adrenergic agonists was analyzed using double mutant cycles. Binding affinities for catecholic and noncatecholic agonists were measured in wild-type and mutant receptors, carrying alanine replacement of the two serines (S204A, S207A β₂-AR), a constitutive activating mutation, or both. The free energy coupling between the losses of binding energy attributable to OH deletion from the ligand and from the receptor indicates a strong interaction (nonadditivity) as expected for a direct binding between the two sets of groups. However, we also measured a significant interaction between the deletion of OH groups from the receptor and the constitutive activating mutation. This suggests that a fraction of the decrease in agonist affinity caused by serine mutagenesis may involve a shift in the conformational equilibrium of the receptor toward the inactive state. Direct measurements using a transient transfection assay confirm this prediction. The constitutive activity of the (S204A, S207A) β₂-AR mutant is 50 to 60% lower than that of the wild-type β₂-AR. We conclude that S204 and S207 do not only provide a docking site for the agonist, but also control the equilibrium of the receptor between active (R*) and inactive (R) forms.