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.

The lipid composition of cell membranes has increasingly been recognized as playing an important role in the function of various membrane proteins, including G Protein-Coupled Receptors (GPCRs). For instance, experimental and computational evidence has pointed to lipids influencing receptor oligomerization directly, by physically interacting with the receptor, and/or indirectly, by altering the bulk properties of the membrane. While the exact role of oligomerization in the function of class A GPCRs such as the μ-opioid receptor (MOR) is still unclear, insight as to how these receptors oligomerize and the relevance of the lipid environment to this phenomenon is crucial to our understanding of receptor function. To examine the effect of lipids and different MOR conformations on receptor oligomerization we carried out extensive coarse-grained molecular dynamics simulations of crystal structures of inactive and/or activated MOR embedded in an idealized mammalian plasma membrane composed of 63 lipid types asymmetrically distributed across the two leaflets. The results of these simulations point, for the first time, to specific direct and indirect effects of the lipids, as well as the receptor conformation, on the spatio-temporal organization of MOR in the plasma membrane. While sphingomyelin-rich, high-order lipid regions near certain transmembrane (TM) helices of MOR induce an effective long-range attractive force on individual protomers, both long-range lipid order and interface formation are found to be conformation dependent, with a larger number of different interfaces formed by inactive MOR compared to active MOR.

Substantial evidence in support of the formation of opioid receptor (OR) di-/oligomers suggests previously unknown mechanisms used by these proteins to exert their biological functions. In an attempt to guide experimental assessment of the identity of the minimal signaling unit for ORs, we conducted extensive coarse-grained (CG) molecular dynamics (MD) simulations of different combinations of the three major OR subtypes, i.e., μ-OR, δ-OR, and κ-OR, in an explicit lipid bilayer. Specifically, we ran multiple, independent MD simulations of each homomeric μ-OR/μ-OR, δ-OR/δ-OR, and κ-OR/κ-OR complex, as well as two of the most studied heteromeric complexes, i.e., δ-OR/μ-OR and δ-OR/κ-OR, to derive the preferred supramolecular organization and dimer interfaces of ORs in a cell membrane model. These simulations yielded over 250 microseconds of accumulated data, which correspond to approximately 1 millisecond of effective simulated dynamics according to established scaling factors of the CG model we employed. Analysis of these data indicates similar preferred supramolecular organization and dimer interfaces of ORs across the different receptor subtypes, but also important differences in the kinetics of receptor association at specific dimer interfaces. We also investigated the kinetic properties of interfacial lipids, and explored their possible role in modulating the rate of receptor association and in promoting the formation of filiform aggregates, thus supporting a distinctive role of the membrane in OR oligomerization and, possibly, signaling.

Considerable evidence has accumulated in recent years suggesting that G protein-coupled receptors (GPCRs) associate in the plasma membrane to form homo- and/or heteromers. Nevertheless, the stoichiometry, fraction and lifetime of such receptor complexes in living cells remain topics of intense debate. Motivated by experimental data suggesting differing stabilities for homomers of the cognate human β1- and β2-adrenergic receptors, we have carried out approximately 160 microseconds of biased molecular dynamics simulations to calculate the dimerization free energy of crystal structure-based models of these receptors, interacting at two interfaces that have often been implicated in GPCR association under physiological conditions. Specifically, results are presented for simulations of coarse-grained (MARTINI-based) and atomistic representations of each receptor, in homodimeric configurations with either transmembrane helices TM1/H8 or TM4/3 at the interface, in an explicit lipid bilayer. Our results support a definite contribution to the relative stability of GPCR dimers from both interface sequence and configuration. We conclude that β1- and β2-adrenergic receptor homodimers with TM1/H8 at the interface are more stable than those involving TM4/3, and that this might be reconciled with experimental studies by considering a model of oligomerization in which more stable TM1 homodimers diffuse through the membrane, transiently interacting with other protomers at interfaces involving other TM helices.

Extensive experimental information supports the formation of ligand-specific conformations of G protein-coupled receptors (GPCRs) as a possible molecular basis for their functional selectivity for signaling pathways. Taking advantage of the recently published inactive and active crystal structures of GPCRs, we have implemented an all-atom computational strategy that combines different adaptive biasing techniques to identify ligand-specific conformations along pre-determined activation pathways. Using the prototypic GPCR β2-adrenergic receptor as a suitable test case for validation, we show that ligands with different efficacies (either inverse agonists, neutral antagonists, or agonists) modulate the free-energy landscape of the receptor by shifting the conformational equilibrium towards active or inactive conformations depending on their elicited physiological response. Notably, we provide for the first time a quantitative description of the thermodynamics of the receptor in an explicit atomistic environment, which accounts for the receptor basal activity and the stabilization of different active-like states by differently potent agonists. Structural inspection of these metastable states reveals unique conformations of the receptor that may have been difficult to retrieve experimentally.

SUMMARY: Rapid expansion of available data about G Protein Coupled Receptor (GPCR) dimers/oligomers over the past few years requires an effective system to organize this information electronically. Based on an ontology derived from a community dialog involving colleagues using experimental and computational methodologies, we developed the GPCR-Oligomerization Knowledge Base (GPCR-OKB). GPCR-OKB is a system that supports browsing and searching for GPCR oligomer data. Such data were manually derived from the literature. While focused on GPCR oligomers, GPCR-OKB is seamlessly connected to GPCRDB, facilitating the correlation of information about GPCR protomers and oligomers. AVAILABILITY AND IMPLEMENTATION: The GPCR-OKB web application is freely available at http://www.gpcr-okb.org

BACKGROUND: G Protein-Coupled Receptors (GPCRs) are a large and diverse family of membrane proteins whose members participate in the regulation of most cellular and physiological processes and therefore represent key pharmacological targets. Although several bioinformatics resources support research on GPCRs, most of these have been designed based on the traditional assumption that monomeric GPCRs constitute the functional receptor unit. The increase in the frequency and number of reports about GPCR dimerization/oligomerization and the implication of oligomerization in receptor function makes necessary the ability to store and access information about GPCR dimers/oligomers electronically. RESULTS: We present here the requirements and ontology (the information scheme to describe oligomers and associated concepts and their relationships) for an information system that can manage the elements of information needed to describe comprehensively the phenomena of both homo- and hetero-oligomerization of GPCRs. The comprehensive information management scheme that we plan to use for the development of an intuitive and user-friendly GPCR-Oligomerization Knowledge Base (GPCR-OKB) is the result of a community dialog involving experimental and computational colleagues working on GPCRs. CONCLUSION: Our long term goal is to disseminate to the scientific community organized, curated, and detailed information about GPCR dimerization/oligomerization and its related structural context. This information will be reported as close to the data as possible so the user can make his own judgment on the conclusions drawn for a particular study. The requirements and ontology described here will facilitate the development of future information systems for GPCR oligomers that contain both computational and experimental information about GPCR oligomerization. This information is freely accessible at http://www.gpcr-okb.org.