Induced-fit mechanism for catecholamine binding to the β2-adrenergic receptor

We engineered single and multiple mutations of serines 203, 204, and 207 in the fifth transmembrane domain of the beta(2)-adrenergic receptor, a region known to interact with hydroxyl groups of the catechol ring. Using such mutants, we measured the binding affinities of a panel of six catecholamine agonists differing only in the presence of substituents in the ethanolamine tail of the molecule. Although all ligands shared an intact catechol ring, they exhibited different losses of binding energy in response to the mutations. For all mutations, we found a clear relationship between the loss of binding caused by receptor mutation and that caused by the ligand modification. This indicates that the catechol ring and the ethanolamine tail synergistically influence their respective interactions when binding to the receptor. To verify this idea by a formal thermodynamic test, we used a double-mutant cycle analysis. We compared the effects of each receptor mutation with those induced by the modifications of the ligand's tail. Because such changes disrupt interactions occurring at different receptor domains, they should produce cumulative losses. In contrast, we found positive cooperativity between such effects. This means that the binding of each side of the catecholamine can enhance the binding of the other, through an effect that is probably propagated via a conformational change. We suggest that the agonist-binding pocket is not rigid but is dynamically formed as the ligand builds an increasing number of contacts with the receptor.