Jennifer M. Johnston

Assessing the Relative Stability of Dimer Interfaces in G Protein-Coupled Receptors

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.

Making Structural Sense of Dimerization Interfaces of Delta Opioid Receptor Homodimers

Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed “4” dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed “4/5” dimer) in an explicit lipid−water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.

Membrane Driven Spatial Organization of GPCRs

Spatial organization of G-protein coupled receptors (GPCRs) into dimers and higher order oligomers has been demonstrated in vitro and in vivo. The pharmacological readout was shown to depend on the specific interfaces, but why particular regions of the GPCR structure are involved, and how ligand-determined states change them remains unknown. Here we show why protein-membrane hydrophobic matching is attained upon oligomerization at specific interfaces from an analysis of coarse-grained molecular dynamics simulations of the spontaneous diffusion-interaction of the prototypical beta2-adrenergic (β2AR) receptors in a POPC lipid bilayer. The energy penalty from mismatch is significantly reduced in the spontaneously emerging oligomeric arrays, making the spatial organization of the GPCRs dependent on the pattern of mismatch in the monomer. This mismatch pattern is very different for β2AR compared to the highly homologous and structurally similar β1AR, consonant with experimentally observed oligomerization patterns of β2AR and β1AR. The results provide a mechanistic understanding of the structural context of oligomerization.

Showcasing modern molecular dynamics simulations of membrane proteins through G protein-coupled receptors

Despite many years of dedicated efforts, high-resolution structural determination of membrane proteins lags far behind that of soluble proteins. Computational methods in general, and molecular dynamics (MD) simulations in particular, have represented important alternative resources over the years to advance understanding of membrane protein structure and function. However, it is only recently that much progress has been achieved owing to new high-resolution membrane protein structures, specialized parallel computer architectures, and efficient simulation algorithms. This has definitely been the case for G protein-coupled receptors (GPCRs), which have assumed a leading role in the area of structural biology with several new structures appearing in the literature during the past five years. We provide here a concise overview of recent developments in computational biophysics of membrane proteins, using GPCRs as an example to showcase important information that can be derived from modern MD simulations.

Lessons from Free Energy Simulations of δ-Opioid Receptor Homodimers Involving the Fourth Transmembrane Helix

Several G protein-coupled receptors (GPCRs), including opioid receptors δOR, μOR, and κOR, have been reported to form stable dimers or oligomers in lipid bilayers and cell membranes. This notion has been recently challenged by imaging data supporting a transient nature of GPCR association. Here we use umbrella sampling reconstructed free energies of δOR homodimers involving the fourth transmembrane helix to predict their association constant. The results of these simulations, combined with estimates of diffusion-limited association rates, suggest a short lifetime for δOR homodimers in the membrane, in agreement with recent trends.

Preferred supramolecular organization and dimer interfaces of opioid receptors from simulated self-association.

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.

Differential response to morphine of the oligomeric state of μ-opioid in the presence of δ-opioid receptors.

Prolonged morphine treatment induces extensive desensitization of the μ-opioid receptor (μOR) which is the G-protein-coupled receptor that primarily mediates the cellular response to morphine. To date, the molecular mechanism underlying this process is unknown. Here, we have used live cell fluorescence imaging to investigate whether prolonged morphine treatment affects the physical environment of μOR, or its coupling with G-proteins, in two neuronal cell lines. We find that chronic morphine treatment does not change the amount of enhanced yellow fluorescence protein (eYFP)-tagged μOR on the plasma membrane, and only slightly decreases its association with G-protein subunits. Additionally, morphine treatment does not have a detectable effect on the diffusion coefficient of eYFP-μOR. However, in the presence of another family member, the δ-opioid receptor (δOR), prolonged morphine exposure results in a significant increase in the diffusion rate of μOR. Number and brightness measurements suggest that μOR exists primarily as a dimer that will oligomerize with δOR into tetramers, and morphine promotes the dissociation of these tetramers. To provide a plausible structural context to these data, we used homology modeling techniques to generate putative configurations of μOR-δOR tetramers. Overall, our studies provide a possible rationale for morphine sensitivity.

Differential stability of the crystallographic interfaces of mu- and kappa-opioid receptors.

The recent mu-opioid receptor (MOPr) and kappa-opioid receptor (KOPr) crystal structures have inspired hypotheses of physiologically relevant dimerization contacts, specifically: a closely packed interface involving transmembrane (TM) helices TM5 and TM6, and a less compact interface, involving TM1, TM2, and helix 8 (H8). While the former was only found in MOPr crystals, similar arrangements of the latter were identified for both KOPr and MOPr. The relevance of these interfaces outside of a crystal lattice is called into question by the possibility that they might be influenced by the specific crystallization conditions. In this study, we have employed umbrella sampling molecular dynamics simulations of coarse-grained representations of the interacting MOPr or KOPr crystallographic structures, in the absence of the T4 lysozyme, and in an explicit lipid-water environment, to determine the strength of receptor dimerization at the different crystallographic interfaces. We note that the shape of the interface plays a dominant role in the strength of the interaction, and the pattern of contacting residues defines the shape of the potential of mean force. This information can be used to guide experiments aimed at exploring the role of dimerization in opioid receptor function.

Beyond Standard Molecular Dynamics: Investigating the Molecular Mechanisms of G Protein-Coupled Receptors with Enhanced Molecular Dynamics Methods

The majority of biological processes mediated by G Protein-Coupled Receptors (GPCRs) take place on timescales that are not conveniently accessible to standard molecular dynamics (MD) approaches, notwithstanding the current availability of specialized parallel computer architectures, and efficient simulation algorithms. Enhanced MD-based methods have started to assume an important role in the study of the rugged energy landscape of GPCRs by providing mechanistic details of complex receptor processes such as ligand recognition, activation, and oligomerization. We provide here an overview of these methods in their most recent application to the field.