Ligand Stabilized Conformational States of Human 2 Adrenergic Receptor: Insight into G Protein Coupled Receptor Activation

¹ Beckman Research Institute, City of Hope Medical Center
² Beckman Research Institute of the City of Hope

^* To whom correspondence should be addressed. E-mail: nvaidehi{at}coh.org.

Submitted on July 17, 2007
Revised on September 21, 2007
Accepted on 12 November 2007

	Abstract

G-protein coupled receptors (GPCRs) are known to exist in dynamic equilibrium between inactive and several active state conformations even in the absence of a ligand. Recent experimental studies on the ₂ Adrenergic Receptor (₂AR) indicate that, structurally different ligands with varying efficacies trigger distinct conformational changes and stabilize different receptor conformations. We have developed a computational method to study the ligand induced rotational orientation changes in the transmembrane helices of GPCRs. This method involves a systematic spanning of the rotational orientation of the transmembrane (TM) helices that are in the vicinity of the ligand for predicting the helical rotations that occur on ligand binding. The predicted ligand stabilized receptor conformations are characterized by a simultaneous lowering of the ligand binding energy and a significant gain in inter-helical and receptor-ligand hydrogen bonds. Using the ₂AR as a model, we show that the receptor conformational state depends on the structure and the efficacy of the ligand for a given signaling pathway. We have studied the ligand stabilized receptor conformations of five different ligands, a full agonist norepinephrine, a partial agonist salbutamol, a weak partial agonist dopamine, a very weak agonist catechol and an inverse agonist ICI-115881. The predicted ligand stabilized receptor models correlate well with the experimentally observed conformational switches in ₂AR, namely the breaking of the ionic lock between R131^3.50 at the intracellular end of TM3 (part of DRY motif) and E268^6.30 on TM6, and the rotamer toggle switch on W286^6.48 on TM6. In agreement with trp-bimane quenching experiments we find that norepinephrine and dopamine break the ionic lock and engage the rotamer toggle switch, whereas salbutamol, a non-catechol partial agonist only breaks the ionic lock and the weak agonist catechol only engages the rotamer toggle switch. Norepinephrine and dopamine occupy the same binding region between TMs 3, 5 and 6, while the binding site of salbutamol is shifted towards TM4. Catechol binds deeper into the protein cavity compared to the other ligands, making contact with TM5 and 6. A part of the catechol binding site overlaps with those of dopamine and norepinephrine but not with that of salbutamol. Virtual Ligand screening on 10,060 ligands on the norepinephrine stabilized receptor conformation shows an enrichment of 38% compared to ligand unbound receptor conformation. These results show that ligand induced conformational changes are important to account for developing functionally specific drugs that will stabilize a particular receptor conformation. These studies represent the first step towards a more universally applicable computational method for studying ligand efficacy and GPCR activation.

Key Words: GPCR, activation, adrenergic, conformational change, drug design, norepinephrine