Structural determinants of the supramolecular organization of G protein-coupled receptors in bilayers

The G protein-coupled receptor (GPCR) rhodopsin self-assembles into supramolecular structures in native bilayers, but the structural determinants of receptor oligomerization are not known. We carried out multiple self-assembly coarse-grained molecular dynamics (CGMD) simulations of model membranes containing up to 64 molecules of the visual receptor rhodopsin over time scales reaching 100 μs. The simulations show strong preferential interaction modes between receptors. Two primary modes of receptor-receptor interactions are consistent with umbrella sampling/potential of mean force (PMF) calculations as a function of the distance between a pair of receptors. The preferential interfaces, involving helices (H) 1/8, 4/5 and 5, present no energy barrier to forming a very stable receptor dimer. Most notably, the PMFs show that the preferred rhodopsin dimer exists in a tail-to-tail conformation, with the interface comprising transmembrane H1/H2 and amphipathic H8 at the extracellular and cytoplasmic surfaces, respectively. This dimer orientation is in line with earlier electron microscopy, X-ray, and cross-linking experiments of rhodopsin and other GPCRs. Less stable interfaces, involving H4 and H6, have a free energy barrier for desolvation (delipidation) of the interfaces and appear to be designed to stabilize "lubricated" (i.e., lipid-coated) dimers. The overall CGMD strategy used here is general and can be applied to study the homo- and heterodimerization of GPCRs and other transmembrane proteins. Systematic extension of the work will deepen our understanding of the forces involved in the membrane organization of integral membrane proteins.     

Molecular dynamics simulations of lipid bilayers

Computer simulation methods are becoming increasingly widespread as tools for studying the structure and dynamics of lipid bilayer membranes. The length scale and time scale accessible to atomic-level molecular dynamics simulations are rapidly increasing, providing insight into the relatively slow motions of molecular reorientation and translation and demonstrating that effects due to the finite size of the simulation cell can influence simulation results. Additionally, significant advances have been made in the complexity of membrane systems studied, including bilayers with cholesterol, small solute molecules, and lipid-protein and lipid-DNA complexes. Especially promising is the progress that continues to be made in the comparison of simulation results with experiment, both to validate the simulation algorithms and to aid in the interpretation of existing experimental data.     

Molecular dynamics simulations of cardiolipin bilayers

Cardiolipin is a key lipid component in the inner mitochondrial membrane, where the lipid is involved in energy production, cristae structure, and mechanisms in the apoptotic pathway. In this article we used molecular dynamics computer simulations to investigate cardiolipin and its effect on the structure of lipid bilayers. Three cardiolipin/POPC bilayers with different lipid compositions were simulated: 100, 9.2, and 0% cardiolipin. We found strong association of sodium counterions to the carbonyl groups of both lipid types, leaving in the case of 9.2% cardiolipin virtually no ions in the aqueous compartment. Although binding occurred primarily at the carbonyl position, there was a preference to bind to the carbonyl groups of cardiolipin. Ion binding and the small headgroup of cardiolipin gave a strong ordering of the hydrocarbon chains. We found significant effects in the water dipole orientation and water dipole potential which can compensate for the electrostatic repulsion that otherwise should force charged lipids apart. Several parameters relevant for the molecular structure of cardiolipin were calculated and compared with results from analyses of coarse-grained simulations and available X-ray structural data.     

Biophysical approaches to G protein-coupled receptors: Structure, function and dynamics

G protein-coupled receptors (GPCR) represent a large family of drug targets for which there is no high-resolution structural information. In order to understand the mechanisms of ligand recognition and receptor activation, there is a strong need for novel biophysical methods. In this Perspective we provide an overview of recent experimental approaches used to explore the molecular architecture and dynamics of GPCR and their interactions with ligands and G proteins using biophysical, non-crystallographic, methods.     

Biomimetic screening of class-B G protein-coupled receptors

The 41-amino acid peptide corticotropin releasing factor (CRF) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the corticotropin releasing factor receptor 1 (CRF(1)R), a typical member of the class-B G-protein-coupled receptors (GPCRs) and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRF with the transmembrane domain of its cognate receptor, we developed a high-throughput conjugation approach that mimics the natural activation mechanism of class-B GPCRs. An acetylene-tagged peptide library was synthesized and conjugated to an azide-modified high-affinity carrier peptide derived from the CRF C-terminus using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstituted potent agonists and were tested in situ for activation of the CRF(1) receptor in a cell-based assay. By use of this approach we (i) defined the minimal sequence motif that is required for full receptor activation, (ii) identified the critical functional groups and structure-activity relationships, (iii) developed an optimized, highly modified peptide probe with high potency (EC(50) = 4 nM) that is specific for the activation domain of the receptor, and (iv) probed the behavioral role of CRF receptors in living mice. The membrane recruitment by a high-affinity carrier enhanced the potency of the tethered peptides by >4 orders of magnitude and thus allowed the testing of very weak initial fragments that otherwise would have been inactive on their own. As no chromatography purification of the test peptides was necessary, a substantial increase in screening throughput was achieved. Importantly, the peptide conjugates can be used to probe the endogenous receptor in its native environment in vivo.     

Improved dissipative particle dynamics simulations of lipid bilayers

The authors introduce a new parameterization for the dissipative particle dynamics simulations of lipid bilayers. In this parameterization, the conservative pairwise forces between beads of the same type in two different hydrophobic chains are chosen to be less repulsive than the water-water interaction, but the intrachain bead interactions are the same as the water-water interaction. For a certain range of parameters, the new bilayer can only be stretched up to 30% before it ruptures. Membrane tension, density profiles, and the in-plane lipid diffusion coefficient of the new bilayer are discussed in detail. They find two kinds of finite size effects that influence the membrane tension: lateral finite size effects, for which larger membranes rupture at a smaller stretch, and transverse finite size effects, for which tensionless bilayers are more compact in larger systems. These finite size effects become rather small when the simulation box is sufficiently large.     

Molecular dynamics simulations of PAMAM dendrimer-induced pore formation in DPPC bilayers with a coarse-grained model

We have performed 0.5-micros-long molecular dynamics (MD) simulations of 0%, 50%, and 100% acetylated third- (G3) and fifth-generation (G5) polyamidoamine (PAMAM) dendrimers in dipalmitoylphosphatidylcholine (DPPC) bilayers with explicit water using the coarse-grained (CG) model developed by Marrink et al. (J.Phys. Chem. B 2004, 108, 750-760), but with long-range electrostatic interactions included. Radii of gyration of the CG G5 dendrimers are 1.99-2.32 nm, close to those measured in the experiments by Prosa et al. (J. Polym. Sci. 1997, 35, 2913-2924) and atomistic simulations by Lee et al. (J. Phys. Chem. B 2006, 110, 4014-4019). Starting with the dendrimer initially positioned near the bilayer, we find that positively charged un-acetylated G3 and 50%-acetylated and un-acetylated G5 dendrimers insert themselves into the bilayer, and only un-acetylated G5 dendrimer induces hole formation at 310 K, but not at 277 K, which agrees qualitatively with experimental observations of Hong et al. (Bioconj. Chem. 2004, 15, 774-782) and Mecke et al. (Langmuir 2005, 21, 10348-10354). At higher salt concentration (approximately 500 mM NaCl), un-acetylated G5 dendrimer does not insert into the bilayer. The results suggest that with inclusion of long-range electrostatic interactions into coarse-grained models, realistic MD simulation of membrane-disrupting effects of nanoparticles at the microsecond time scale is now possible.     

Molecular dynamics simulations of rhodopsin in different one-component lipid bilayers

Four 20 ns molecular dynamic simulations of rhodopsin embedded in different one-component lipid bilayers have been carried out to ascertain the importance of membrane lipids on the protein structure. Specifically, dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl linoleyl phosphatidylcholine (PLPC) lipid bilayers have been considered for the present work. The results reported here provide information on the hydrophobic matching between the protein and the bilayer and about the differential effects of the protein on the thickness of the different membranes. Furthermore, a careful analysis of the individual protein-lipid interactions permits the identification of residues that exhibit permanent interactions with atoms of the lipid environment that may putatively act as hooks of the protein to the membrane. The analysis of the trajectories also provides information about the effect of the bilayer on the protein structure, including secondary structural elements, salt bridges, and rigid-body motions.     

Latest development in drug discovery on G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the family of proteins with the highest impact from social, therapeutic and economic point of view. Today, more than 50% of drug targets are based on GPCRs and the annual worldwide sales exceeds 50 billion dollars. GPCRs are involved in all major disease areas such as cardiovascular, metabolic, neurodegenerative, psychiatric, cancer and infectious diseases. The classical drug discovery process has relied on screening compounds, which interact favorably with the GPCR of interest followed by further chemical engineering as a mean of improving efficacy and selectivity. In this review, methods for sophisticated chemical library screening procedures will be presented. Furthermore, development of cell-based assays for functional coupling of GPCRs to G proteins will be discussed. Finally, the possibility of applying structure-based drug design will be summarized. This includes the application of bioinformatics knowledge and molecular modeling approaches in drug development programs. The major efforts established through large networks of structural genomics on GPCRs, where recombinantly expressed GPCRs are subjected to purification and crystallization attempts with the intention of obtaining high-resolution structures, are presented as a promising future approach for tailor-made drug development.     

Artifacts in dynamical simulations of coarse-grained model lipid bilayers

With special focus on dissipative particle dynamics simulations of anisotropic and complex soft matter, such as lipid bilayers in water, we have investigated the occurrence of artifacts in the results obtained from dynamical simulations of coarse-grained particle-based models. The particles are modeled by beads that interact via soft repulsive conservative forces (as defined in dissipative particle dynamics simulations), harmonic bond potentials, as well as bending potentials imparting stiffness to the lipid tails. Two different update schemes are investigated: dissipative particle dynamics with a velocity-Verlet-like integration scheme [G. Besold, I. Vattulainen, M. Karttunen, and J. M. Polson, Phys. Rev. E 63, R7611 (2000)] and Lowe-Andersen thermostatting [C. P. Lowe, Europhys. Lett. 47, 145 (1999)] with the standard velocity-Verlet integration algorithm. By varying the integration time step, we examine various physical quantities, in particular pressure profiles and kinetic bead temperatures, for their sensitivity to artifacts caused by the specific combination of integration technique and the thermostat. We then propose a simple fingerprint method that allows monitoring the presence of simulation artifacts.     

Area compressibility and buckling of amphiphilic bilayers in molecular dynamics simulations

The elastic modulus or area compressibility of a membrane is routinely calculated in molecular dynamics simulations as the proportionality constant relating surface tension and projected surface area. Recent studies, however, have revealed a marked system size dependence of these moduli, which we attribute to the neglect of thermal undulations in the area calculation. We discuss several methods, based on the Helfrich model and on numerical triangulation, to remedy this situation, and find a satisfying agreement between them. The Helfrich model also quantitatively describes a buckling transition observed for compressed bilayers.     

Virtual screen for ligands of orphan G protein-coupled receptors

This paper describes a virtual screening methodology that generates a ranked list of high-binding small molecule ligands for orphan G protein-coupled receptors (oGPCRs), circumventing the requirement for receptor three-dimensional structure determination. Features representing the receptor are based only on physicochemical properties of primary amino acid sequence, and ligand features use the two-dimensional atomic connection topology and atomic properties. An experimental screen comprised nearly 2 million hypothetical oGPCR-ligand complexes, from which it was observed that the top 1.96% predicted affinity scores corresponded to "highly active" ligands against orphan receptors. Results representing predicted high-scoring novel ligands for many oGPCRs are presented here. Validation of the method was carried out in several ways: (1) A random permutation of the structure-activity relationship of the training data was carried out; by comparing test statistic values of the randomized and nonshuffled data, we conclude that the value obtained with nonshuffled data is unlikely to have been encountered by chance. (2) Biological activities linked to the compounds with high cross-target binding affinity were analyzed using computed log-odds from a structure-based program. This information was correlated with literature citations where GPCR-related pathways or processes were linked to the bioactivity in question. (3) Anecdotal, out-of-sample predictions for nicotinic targets and known ligands were performed, with good accuracy in the low-to-high "active" binding range. (4) An out-of-sample consistency check using the commercial antipsychotic drug olanzapine produced "active" to "highly-active" predicted affinities for all oGPCRs in our study, an observation that is consistent with documented findings of cross-target affinity of this compound for many different GPCRs. It is suggested that this virtual screening approach may be used in support of the functional characterization of oGPCRs by identifying potential cognate ligands. Ultimately, this approach may have implications for pharmaceutical therapies to modulate the activity of faulty or disease-related cellular signaling pathways. In addition to application to cell surface receptors, this approach is a generalized strategy for discovery of small molecules that may bind intracellular enzymes and involve protein-protein interactions.     

Investigation of finite system-size effects in molecular dynamics simulations of lipid bilayers

In the absence of external stress, the surface tension of a lipid membrane vanishes at equilibrium, and the membrane exhibits long wavelength undulations that can be described as elastic (as opposed to tension-dominated) deformations. These long wavelength fluctuations are generally suppressed in molecular dynamics simulations of membranes, which have typically been carried out on membrane patches with areas <100 nm2 that are replicated by periodic boundary conditions. As a result, finite system-size effects in molecular dynamics simulations of lipid bilayers have been subject to much discussion in the membrane simulation community for several years, and it has been argued that it is necessary to simulate small membrane patches under tension to properly model the tension-free state of macroscopic membranes. Recent hardware and software advances have made it possible to simulate larger, all-atom systems allowing us to directly address the question of whether the relatively small size of current membrane simulations affects their physical characteristics compared to real macroscopic bilayer systems. In this work, system-size effects on the structure of a DOPC bilayer at 5.4 H2O/lipid are investigated by performing molecular dynamics simulations at constant temperature and isotropic pressure (i.e., vanishing surface tension) of small and large single bilayer patches (72 and 288 lipids, respectively), as well as an explicitly multilamellar system consisting of a stack of five 72-lipid bilayers, all replicated in three dimensions by using periodic boundary conditions. The simulation results are compared to X-ray and neutron diffraction data by using a model-free, reciprocal space approach developed recently in our laboratories. Our analysis demonstrates that finite-size effects are negligible in simulations of DOPC bilayers at low hydration, and suggests that refinements are needed in the simulation force fields.     

Computational prediction of the coupling specificity of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent one of the most important categories of membrane proteins that play important roles in signaling pathways. GPCRs transduce the extracellular stimuli into intracellular second messengers via their coupling to specific class of heterotrimeric GTP-binding proteins (G proteins) and the subsequent regulation of a diverse variety of effectors. Understanding the coupling specificity of GPCRs is critical for further comprehending their function, and is of tremendous clinical significance because GPCRs are the most successful drug targets. This minireview addresses the computational approaches that have been created for the prediction of coupling specificity of GPCRs and highlights the perspective of bioinformatics strategies that may be used to tackle this important task. In addition, some of the important resources of this field are also provided.     

Ligand and decoy sets for docking to G protein-coupled receptors

We compiled a G protein-coupled receptor (GPCR) ligand library (GLL) for 147 targets, selecting for each ligand 39 decoy molecules, collected in the GPCR Decoy Database (GDD). Decoys were chosen ensuring a ligand-decoy similarity of six physical properties, while enforcing ligand-decoy chemical dissimilarity. The performance in docking of the GDD was evaluated on 19 GPCRs, showing a marked decrease in enrichment compared to bias-uncorrected decoy sets. Both the GLL and GDD are freely available for the scientific community.     

Interleaflet interaction and asymmetry in phase separated lipid bilayers: molecular dynamics simulations

In order to investigate experimentally inaccessible, molecular-level detail regarding interleaflet interaction in membranes, we have run an extensive series of coarse-grained molecular dynamics simulations of phase separated lipid bilayers. The simulations are motivated by differences in lipid and cholesterol composition in the inner and outer leaflets of biological membranes. Over the past several years, this phenomenon has inspired a series of experiments in model membrane systems which have explored the effects of lipid compositional asymmetry in the two leaflets. The simulations are directed at understanding one potential consequence of compositional asymmetry, that being regions of bilayers where liquid-ordered (L(o)) domains in one leaflet are opposite liquid-disordered (L(d)) domains in the other leaflet (phase asymmetry). The simulated bilayers are of two sorts: 1) Compositionally symmetric leaflets where each of the two leaflets contains an identical, phase separated (L(o)/L(d)) mixture of cholesterol, saturated and unsaturated phospholipid; and 2) Compositionally asymmetric leaflets, where one leaflet contains a phase separated (L(o)/L(d)) mixture while the other contains only unsaturated lipid, which on its own would be in the L(d) phase. In addition, we have run simulations where the lengths of the saturated lipid chains as well as the mole ratios of the three lipid components are varied. Collectively, we report on three types of interleaflet coupling within a bilayer. First, we show the effects of compositional asymmetry on acyl chain tilt and order, lipid rotational dynamics, and lateral diffusion in regions of leaflets that are opposite L(o) domains. Second, we show substantial effects of compositional asymmetry on local bilayer curvature, with the conclusion that phase separated leaflets resist curvature, while inducing large degrees of curvature in an opposing L(d) leaflet. Finally, in compositionally symmetric, phase separated bilayers, we find phase asymmetry (domain antiregistration) between the two leaflets occurs as a consequence of mismatched acyl chain-lengths in the saturated and unsaturated lipids.     

Photo-affinity labeling strategy to study the binding site of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are involved in the control of every aspect of our behavior and physiology. GPCR can be involved in pathological processes as well and are linked to numerous diseases, including cardiovascular and mental disorders, retinal degeneration, cancer, and AIDS. This article reviews the methods of approaching photo-affinity labeling strategy to obtain the possible G protein-coupled receptors’s binding site.     

Population dynamics simulations of functional model proteins

In order to probe the fundamental principles that govern protein evolution, we use a minimalist model of proteins to provide a mapping from genotype to phenotype. The model is based on physically realistic forces of protein folding and includes an explicit definition of protein function. Thus, we can find the fitness of a sequence from its ability to fold to a stable structure and perform a function. We study the fitness landscapes of these functional model proteins, that is, the set of all sequences mapped on to their corresponding fitnesses and connected to their one mutant neighbors. Through population dynamics simulations we directly study the influence of the nature of the fitness landscape on evolution. Populations are observed to move to a steady state, the distribution of which can often be predicted prior to the population dynamics simulations from the nature of the fitness landscape and a quantity analogous to a partition function. In this paper, we develop a scheme for predicting the steady-state population on a fitness landscape, based on the nature of the fitness landscape, thereby obviating the need for explicit population dynamics simulations and providing some insight into the impact on molecular evolution of the nature of fitness landscapes. Poor predictions are indicative of fitness landscapes that consist of a series of weakly connected sublandscapes.