Kinetic and thermodynamic insights into sodium ion translocation through the μ-opioid receptor from molecular dynamics and machine learning analysis

Abstract

The differential modulation of agonist and antagonist binding to opioid receptors (ORs) by sodium (Na+) has been known for decades. To shed light on the molecular determinants, thermodynamics, and kinetics of Na+ translocation through the μ-OR (MOR), we used a multi-ensemble Markov model framework combining equilibrium and non-equilibrium atomistic molecular dynamics simulations of Na+ binding to MOR active or inactive crystal structures embedded in an explicit lipid bilayer. We identify an energetically favorable, continuous ion pathway through the MOR active conformation only, and provide, for the first time: i) estimates of the energy differences and required timescales of Na+ translocation in inactive and active MORs, ii) estimates of Na+-induced changes to agonist binding validated by radioligand measurements, and iii) testable hypotheses of molecular determinants and correlated motions involved in this translocation, which are likely to play a key role in MOR signaling.

Harnessing Ion-Binding Sites for GPCR Pharmacology

“…pH Dependence. Protonation of D 2.50 has been proposed as a facilitator of Na 1 egress from class A GPCRs, thus shifting the conformational equilibrium toward their active state and facilitating signaling (Vickery et al, 2018;Hu et al, 2019). This mechanism is consistent with in vitro observations that lower pH increases both basal and ligand-induced activation, for example in the b 2 AR (Ghanouni et al, 2000).…”

Mechanism of µ-Opioid Receptor-Magnesium Interaction and Positive Allosteric Modulation

“…As expected, in the ligand-free inactive MOP receptor system (Figure 1), the Na + cation has the highest affinity for the allosteric binding site (in particular, residues D114 2.50 , N150 3.35 , S154 3.39 , N328 7.45 , and W293 6.48 ) revealed by high-resolution crystal structures of various GPCRs. We calculate a = 0.05 M for Na + at this site (Figure 1e), which agrees with our previously published calculations from simulations of the MOP receptor in the presence of Na + only, and their experimental validation (41). In addition to the crystallographic binding site, the Na + cation was also found to bind at an extracellular site (specifically, residues N127 2.63 , D216 ECL2 , C217 ECL2 , T218 ECL2 ; see Figure 1d) in the inactive MOP receptor system, albeit with the significantly lower affinity of 3.4 M. Unlike Na + , Mg 2+ ions bound primarily at three slightly different sites on the extracellular region of the inactive MOP receptor system, which all had the D216 ECL2 residue coordinating the cation (Figure 1a (Figure 2d).…”

“…Indeed, it has been known for more than forty years that the MOP receptor can be differentially modulated by cations (8). While the monovalent Na + cation can decrease agonist affinity at the MOP receptor (8), most likely though stabilization of the inactive conformational state of the receptor (e.g., see (9,10)), the divalent Mg 2+ cation has the opposite effect (e.g., see (11)), suggesting it stabilizes an active-like conformation of the receptor. Notably, similar conclusions were drawn for other GPCRs based on inferences from biochemical and pharmacological studies (12)(13)(14)(15)(16)(17)(18).…”

“…While various high-resolution X-ray crystal structures of inactive forms of GPCRs have revealed the atomic details of Na + binding (19)(20)(21)(22), and several MD simulation studies have supported the stabilization of inactive conformations of the receptor by this monovalent cation (e.g., see (9,10,23,24)), limited information exists to date about the preferred binding site(s) of Mg 2+ and the molecular mechanism underlying the potential positive allosteric effect of this divalent cation. A recent interdisciplinary study combining the results of 19 F-NMR and MD simulations on another prototypic GPCR, the adenosine A2A receptor (18), provided further support to the negative allosteric modulation of receptors by Na + and their positive allosteric modulation by Ca 2+ and Mg 2+ .…”

Opioid Pharmacology under the Microscope

“…Moreover, there is accumulating evidence for additional factors impacting bias profile, particularly kinetic effects on trafficking and signaling (Thompson et al, 2016;Weinberg et al, 2017). In the future, we anticipate that structural, biophysical and computational approaches will provide increasingly precise understanding of the underpinnings of agonist efficacy, bias and allosteric modulation leading to drugs with improved therapeutic window (Filizola, 2019;Hu, Wang, et al, 2019;Zarzycka et al, 2019).…”