Antagonist conformations within the β₂-adrenergic receptor ligand binding pocket

The interactions between β-adrenergic receptor (βAR) antagonists and the β₂AR were studied with the use of photoaffinity labels. A proteolytic map of the receptor was made and confirmed through amino-terminal amino acid sequencing by locating sites of derivatization. [¹²⁵I]lodoazidothiophenylalprenolol (IAPTA) is a photoaffinity derivative of the βAR antagonist alprenolol with a photoactivatable group on the aryloxy end of the molecule. IAPTA exclusively derivatizes a peptide consisting of transmembrane domains (TMs) 6 and 7 of the hamster lung β₂AR, supporting the contention that TMs 6 and 7 interact with the aryloxy portion of the βAR antagonist pharmacophore. The βAR antagonist photoaffinity labels [¹²⁵I]iodoazidobenzylpindolol (IABP), [¹²⁵I]iodoazidophenyl CGP- 12177A (IAPCGP), and [¹²⁵I]iodocyanopindololdiazarene (ICYPdz) are similar in that their photoactive moieties are attached to the amino end of the antagonist pharmacophore. IABP derivatized TMs 5-7 and a peptide containing TM 1 to approximately equal extents. IAPCGP derivatized TMs 6 and 7 >> TM 5 = TM 4 = TMs 2 and 3 = TM 1. ICYPdz derivatized TM 1 >> TMs 6 and 7 > TM 4. We conclude that the aryloxy end of the βAR antagonist pharmacophore is highly constrained within TMs 6 and 7, whereas the amino terminus is much less constrained and able to assume multiple conformations. Molecular dynamics simulations predict that IABP, IAPCGP, and ICYPdz favor a folded conformation, with both ends close together. Derivatization of TMs 6 and 7 by IABP, IAPCGP, and ICYPdz suggests the folded conformation of these compounds in the ligand binding pocket.