Quaternary structure of a G-protein coupled receptor heterotetramer in complex with Gi and Gs

Background: G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. Results: We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. Conclusions: The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function

Adenosine/A2B receptor signaling ameliorates the effects of ageing and counteracts obesity

The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential

Identifying human diamine sensors for death related putrescine and cadaverine molecules

Pungent chemical compounds originating from decaying tissue are strong drivers of animal behavior. Two of the best-characterized death smell components are putrescine (PUT) and cadaverine (CAD), foul-smelling molecules produced by decarboxylation of amino acids during decomposition. These volatile polyamines act as 'necromones', triggering avoidance or attractive responses, which are fundamental for the survival of a wide range of species. The few studies that have attempted to identify the cognate receptors for these molecules have suggested the involvement of the seven-helix trace amine-associated receptors (TAARs), localized in the olfactory epithelium. However, very little is known about the precise chemosensory receptors that sense these compounds in the majority of organisms and the molecular basis of their interactions. In this work, we have used computational strategies to characterize the binding between PUT and CAD with the TAAR6 and TAAR8 human receptors. Sequence analysis, homology modeling, docking and molecular dynamics studies suggest a tandem of negatively charged aspartates in the binding pocket of these receptors which are likely to be involved in the recognition of these small biogenic diamines

Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs

Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs

Molecular dynamics simulation studies of the interactions between ionic liquids and amino acids in aqueous solution

Although the understanding of the influence of ionic liquids (ILs) on the solubility behavior of biomolecules in aqueous solutions is relevant for the design and optimization of novel biotechnological processes, the underlying molecular-level mechanisms are not yet consensual or clearly elucidated. In order to contribute to the understanding of the molecular interactions established between amino acids and ILs in aqueous media, classical molecular dynamics (MD) simulations were performed for aqueous solutions of five amino acids with different structural characteristics (glycine, alanine, valine, isoleucine, and glutamic acid) in the presence of 1-butyl-3-methylimidazolium bis(trifluoromethyl)sulfonyl imide. The results from MD simulations enable to relate the properties of the amino acids, namely their hydrophobicity, to the type and strength of their interactions with ILs in aqueous solutions and provide an explanation for the direction and magnitude of the solubility phenomena observed in [IL + amino acid + water] systems by a mechanism governed by a balance between competitive interactions of the IL cation, IL anion, and water with the amino acids

The ELBA Force Field for Coarse-Grain Modeling of Lipid Membranes

A new coarse-grain model for molecular dynamics simulation of lipid membranes is presented. Following a simple and conventional approach, lipid molecules are modeled by spherical sites, each representing a group of several atoms. In contrast to common coarse-grain methods, two original (interdependent) features are here adopted. First, the main electrostatics are modeled explicitly by charges and dipoles, which interact realistically through a relative dielectric constant of unity (). Second, water molecules are represented individually through a new parametrization of the simple Stockmayer potential for polar fluids; each water molecule is therefore described by a single spherical site embedded with a point dipole. The force field is shown to accurately reproduce the main physical properties of single-species phospholipid bilayers comprising dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE) in the liquid crystal phase, as well as distearoylphosphatidylcholine (DSPC) in the liquid crystal and gel phases. Insights are presented into fundamental properties and phenomena that can be difficult or impossible to study with alternative computational or experimental methods. For example, we investigate the internal pressure distribution, dipole potential, lipid diffusion, and spontaneous self-assembly. Simulations lasting up to 1.5 microseconds were conducted for systems of different sizes (128, 512 and 1058 lipids); this also allowed us to identify size-dependent artifacts that are expected to affect membrane simulations in general. Future extensions and applications are discussed, particularly in relation to the methodology's inherent multiscale capabilities