No abstract

Fentanyl is a main driver of the current opioid crisis. As a powerful narcotic, fentanyl initiates biological response through binding to the mu-opioid receptor (mOR); however, the molecular details remain unknown. Here we study the mechanism and predict the kinetics of fentanyl-mOR dissociation by applying an advanced molecular dynamics (MD) technique based on the X-ray structure of the morphinan bound mOR. 144 metadynamics trajectories were performed while accounting for the protonation state of the conserved H29, which has been suggested to modulate ligand-mOR affinity and binding mode. Surprisingly, in some trajectories fentanyl samples a deep pocket 5-10 Angstrom below the level of the conserved D147 before escaping mOR, yielding the calculated residence times of 27 s, 6 s, and 0.6 s, in the presence of the ND-, NE-, and doubly protonated H297, respectively. The former value is within one order of magnitude from the recently measured residence time of about 4 min, suggesting the biological relevance of fentanyl's deep insertion, which is enabled through hydrogen bonding with H297 as well as hydrophobic interactions with transmembrane helix 6. These data support the hypothesis that the molecular mechanism of fentanyl may be distinct from morphinan compounds. Our developed protocol may be used to predict the dissociation rates of other opioids, thereby assisting the evaluation of strategies for drug overdose reversal. Finally, the profound role of the histidine protonation state discovered in this work may shift the paradigm in computational studies of ligand-receptor kinetics.