Event Abstract Back to Event Ligand binding and action of microswitches in G protein coupled receptors Slawomir Filipek1* 1 International Institute of Molecular and Cell Biology, Poland G protein coupled receptors (GPCRs) interact with very diverse sets of ligands which bind to the transmembrane segments and sometimes also to the receptor extracellular domains. Each receptor subfamily undergoes a series of conformational rearrangements leading to the binding of a G protein during the activation process. All GPCRs preserved the 7-TM scaffold during evolution but adapted it to different sets of ligands by structure customization. Binding of structurally different agonists requires the disruption of distinct intramolecular interactions, leading to different receptor conformations and differential effects on downstream signaling proteins. The dynamic character of GPCRs is likely to be essential for their physiological functions, and a better understanding of this molecular plasticity could be important for drug discovery. Experiments suggest that agonist binding and receptor activation occur through a series of conformational intermediates. Transition between these intermediate states involves the disruption of intramolecular interactions that stabilize the basal state of a receptor. Such profound changes are evoked by the action of molecular switches (microswitches). The switches proposed so far for different GPCRs include the “rotamer toggle switch” involving the CWxPxF sequence on helix TM6, the switch based on the NPxxY(x)(5,6)F sequence linking helices TM7 and H8, the “3 7 lock” interaction connecting TM3 and TM7 (involving Schiff base-counterion interaction in rhodopsin), and the “ionic lock” linking transmembrane helices TM3 and TM6 and employing the (E/D)RY motif. To investigate the early activation steps concurrent to ligand binding we used opioid receptors. They belonging to the family A (rhodopsin-like) of GPCRs. For the important role they play in the human body in controlling pain and stress, modulating immune responses and developing addiction the opioid receptors were subject of numerous investigations. We chose a set of rigid ligands with the structural motif of tyramine because ligand flexibility would obscure the very first structural movements induced upon ligand binding. On the basis of conducted molecular dynamics simulations we propose that agonists and antagonists bind to Y3.33 but only agonists are able to move deeper into the receptor binding site and to reach H6.52. The movement from Y3.33 to H6.52 induces breaking of the TM3-TM7 connection D3.32-Y7.43 (“3-7 lock” switch). We also observed a concerted motion of W6.48 and H6.52 suggesting existence of an extended “rotamer toggle switch”. Simultaneous action of both switches, the “3-7 lock” and the “rotamer toggle switch”, implies a temporal but also spatial (an agonist linking H6.52 and D3.32) dependence between them and possibly other switches on a longer time scales.