Abstract
Agonist binding to the extracellular part of G protein-coupled receptors (GPCRs) leads to conformational changes in the transmembrane region that activate cytosolic signalling pathways. Although several high resolution structures of the inactive and active receptor states are available, the atomistic details of the allosteric coupling that transmits the signal across the membrane are not fully understood. We calculated free energy landscapes of β2 adrenergic receptor activation using atomistic molecular dynamics simulations in an optimized string of swarms framework, which sheds new light on the roles of microswitches in governing the equilibrium between conformational states. Contraction of the extracellular binding site in the presence of agonist is obli-gatorily coupled to conformational changes in a connector motif located in the core of the transmembrane region. In turn, the connector is probabilistically coupled to the conformation of the intracellular region: an active connector promotes desolvation of a buried solvent-filled cavity, a twist of the conserved NPxxY motif, and an interaction between two conserved tyrosines in transmembrane helices 5 and 7 (Y-Y motif), which leads to a larger population of active-like states at the G protein binding site. This coupling is further augmented by protonation of the strongly conserved Asp792.50, which locks the solvent cavity, NPxxY, and Y-Y motifs in active-like conformations. The agonist binding site hence communicates with the intracellular region via a cascade of locally connected switches and characterizing the free energy landscapes along the conformation of these microswitches contributes to understanding of how ligands can stabilize distinct receptor states. We demonstrate that the developed simulation protocol is transferable to other class A GPCRs and anticipate that it will become a useful tool in the design of drugs with specific signaling properties.