Ions Everywhere? Mg²⁺ in the μ-Opioid GPCR and Atomic Details of Their Impact on Function

Abstract

Filizola and co-workers have applied a combination of long-time molecular dynamics and oscillating chemical potential grand canonical Monte Carlo/molecular dynamics to investigate the distribution of Mg²⁺ and Na⁺ in the μ-opioid receptor and their impact on its function. Results indicate atomic details of potential mechanisms by which Mg²⁺ leads to increased efficacy of opioid analgesics. The presence of information flow between the extracellular loops and the intracellular region of the G-protein-coupled receptors that interacts with G-proteins in the presence of Mg²⁺ may be a phenomenon occurring in other G-protein-coupled receptors and, therefore, potentially of broad impact.

Main Text

Atomic ions are pervasive in biological systems and, although widely studied, there is still a large gap in our knowledge of their role in complex, heterogeneous systems. Filizola and co-workers (1), motivated by experimental data showing that the divalent cation Mg²⁺ leads to increased efficacy of known opioid analgesics (2), potentially through stabilization of active conformation of the G-protein-coupled receptor (GPCR) as well as recent studies on the A_2A-adrenergic GPCR (3), undertook a study of Mg²⁺ and Na⁺ interactions with and impact on the μ-opioid receptor (MOP). A major challenge in studying Mg²⁺ in complex macromolecular systems is the long lifetimes of complexes involving Mg²⁺ ranging from microseconds to milliseconds in the case of water and phosphate, respectively (4). To overcome this, they applied long-time molecular dynamics (ltMD) in conjunction with an enhanced solute sampling technology, oscillating chemical potential grand canonical Monte Carlo/molecular dynamics (osμ_exGCMC/MD) (5). ltMD allows information on the local sampling of ion positions and, importantly, their impact on the conformational dynamics of the system to be obtained, whereas osμ_exGCMC/MD allows for comprehensive sampling of the distribution of ions as well as other small molecular weight solutes to be obtained throughout heterogeneous systems, as previously applied to RNA (6). Filizola and co-workers (1) used this combination in the MOP to allow for sites occupied by Mg²⁺ to be identified with the impact of those occupancies on allostery to be elucidated.

Results from the simulations identify a number of binding sites for Mg²⁺ as well as Na⁺ on both the active and inactive forms of the MOP, with both the charged and neutral states of D^2.50 that occupy the Na⁺ allosteric site investigated. Both ions are observed to bind to extracellular sites and the orthosteric site of the MOP, whereas Na⁺ also interacts with the D^2.50 allosteric site adjacent to the orthosteric site. To investigate the cooperativity of binding of the two ions, the occupancy between different identified binding sites was analyzed. In the majority of cases, negative cooperativity was observed, consistent with occupancy of different sites by similarly charged ions, possibly leading to decreased occupancy by Na⁺ of its allosteric site with Mg²⁺ in the orthosteric site, thereby decreasing the known inhibitory effect of Na⁺ (7). However, in some cases, positive cooperativity was observed. An interesting example was the presence of Na⁺ interacting with a site on extracellular loop 2 (ECL2) leading to enhanced binding of Mg²⁺ at the orthosteric site.

Three phenomena were identified that may contribute to the experimentally observed increased efficacy of MOP agonists in the presence of Mg²⁺. First, the binding of the ion to the active form is favored over the inactive species, indicating the ion may favor the equilibrium toward the active state. Second, occupancy of ECL2 and ECL3 by Mg²⁺ leads to increased sampling of closed states of the loops in the active form of MOP leading to a predicted four- or fivefold increase in agonist binding affinity. Finally, analysis of the impact of Mg²⁺ interactions with ECL2/3 on information flow across the receptor revealed increased flow with the intracellular region of the MOP involved in binding with the G-protein, an interaction required for downstream signaling. Notably, this effect is largest in the active MOP with D^2.50 in the Na⁺ allosteric site in a neutral state, consistent with reports that this is the mostly likely protonation form in the active state.

In summary, the study by Filizola and co-workers (1) yields novel insights into the distribution of cations in the MOP by combining ltMD and osμ_exGCMC/MD technologies. The simulations yield information on the cooperativity between Na⁺ and Mg²⁺ binding, and three phenomena are identified that may contribute to the experimentally observed increase in the efficacy of MOP agonists by Mg²⁺. Of these, the increased information flow between ECL2/3 and the intracellular G-protein binding regions is consistent with observations in the A_2A-adrenergic receptor (3) indicating its potential relevance to other GPCRs. Although novel insights into cation-GPCR interactions were obtained, it is anticipated that closer linkage between the osμ_exGCMC/MD and ltMD technologies will allow for a more comprehensive understanding of the impact of cations on the conformational sampling of complex macromolecules such as GPCRs and RNAs to be achieved. Such information will allow for an expanded atomic detail understanding of the role ions play in affecting important biological functions, a phenomenon that is likely occurring in the large majority of biological macromolecules.

Editor: Vasanthi Jayaraman.