Combination of hypomorphic mutations of the Drosophila homologues of aryl hydrocarbon receptor and nucleosome assembly protein family genes disrupts morphogenesis, memory and detoxification

Aryl hydrocarbon receptor is essential for biological responses to endogenous and exogenous toxins in mammals. Its Drosophila homolog spineless plays an important role in fly morphogenesis. We have previously shown that during morphogenesis spineless genetically interacts with CG5017 gene, which encodes a nucleosome assembly factor and may affect cognitive function of the fly. We now demonstrate synergistic interactions of spineless and CG5017 in pathways controlling oxidative stress response and long-term memory formation in Drosophila melanogaster. Oxidative stress was induced by low doses of X-ray irradiation of flies carrying hypomorphic mutation of spineless, mutation of CG5017, and their combination. To determine the sensitivity of these mutants to pharmacological modifiers of the irradiation effect, we irradiated flies growing on standard medium supplemented by radiosensitizer furazidin and radioprotector serotonin. The effects of irradiation were investigated by analyzing leg and antenna morphological structures and by using real-time PCR to measure mRNA expression levels for spineless, Cyp6g1 and Gst-theta genes. We also examined long-term memory in these mutants using conditioned courtship suppression paradigm. Our results show that the interaction of spineless and CG5017 is important for regulation of morphogenesis, long-term memory formation, and detoxification during oxidative stress. Since spineless and CG5017 are evolutionary conserved, these results must be considered when evaluating the risk of combining similar mutations in other organisms, including humans

Identifying Ligand Binding Conformations of the β2-Adrenergic Receptor by Using Its Agonists as Computational Probes

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β2-adrenergic receptor (β2AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β2AR. We show that the select ligands bind preferentially to different predicted conformers of β2AR, and identify a role of β2AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as "computational probes" to systematically identify protein conformers with likely biological significance. © 2012 Isin et al

Small Molecules with Similar Structures Exhibit Agonist, Neutral Antagonist or Inverse Agonist Activity toward Angiotensin II Type 1 Receptor

Small differences in the chemical structures of ligands can be responsible for agonism, neutral antagonism or inverse agonism toward a G-protein-coupled receptor (GPCR). Although each ligand may stabilize the receptor conformation in a different way, little is known about the precise conformational differences. We synthesized the angiotensin II type 1 receptor blocker (ARB) olmesartan, R239470 and R794847, which induced inverse agonism, antagonism and agonism, respectively, and then investigated the ligand-specific changes in the receptor conformation with respect to stabilization around transmembrane (TM)3. The results of substituted cysteine accessibility mapping studies support the novel concept that ligand-induced changes in the conformation of TM3 play a role in stabilizing GPCR. Although the agonist-, neutral antagonist and inverse agonist-binding sites in the AT1 receptor are similar, each ligand induced specific conformational changes in TM3. In addition, all of the experimental data were obtained with functional receptors in a native membrane environment (in situ)

Ligand-Induced Modulation of the Free-Energy Landscape of G Protein-Coupled Receptors Explored by Adaptive Biasing Techniques

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

Computing Highly Correlated Positions Using Mutual Information and Graph Theory for G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are a superfamily of seven transmembrane-spanning proteins involved in a wide array of physiological functions and are the most common targets of pharmaceuticals. This study aims to identify a cohort or clique of positions that share high mutual information. Using a multiple sequence alignment of the transmembrane (TM) domains, we calculated the mutual information between all inter-TM pairs of aligned positions and ranked the pairs by mutual information. A mutual information graph was constructed with vertices that corresponded to TM positions and edges between vertices were drawn if the mutual information exceeded a threshold of statistical significance. Positions with high degree (i.e. had significant mutual information with a large number of other positions) were found to line a well defined inter-TM ligand binding cavity for class A as well as class C GPCRs. Although the natural ligands of class C receptors bind to their extracellular N-terminal domains, the possibility of modulating their activity through ligands that bind to their helical bundle has been reported. Such positions were not found for class B GPCRs, in agreement with the observation that there are not known ligands that bind within their TM helical bundle. All identified key positions formed a clique within the MI graph of interest. For a subset of class A receptors we also considered the alignment of a portion of the second extracellular loop, and found that the two positions adjacent to the conserved Cys that bridges the loop with the TM3 qualified as key positions. Our algorithm may be useful for localizing topologically conserved regions in other protein families

Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation

The second extracellular loop (EL2) of rhodopsin forms a cap over the binding site of its photoreactive 11-cis retinylidene chromophore. A crucial question has been whether EL2 forms a reversible gate that opens upon activation or acts as a rigid barrier. Distance measurements using solid-state 13C NMR spectroscopy between the retinal chromophore and the β4 strand of EL2 show that the loop is displaced from the retinal binding site upon activation, and there is a rearrangement in the hydrogen-bonding networks connecting EL2 with the extracellular ends of transmembrane helices H4, H5 and H6. NMR measurements further reveal that structural changes in EL2 are coupled to the motion of helix H5 and breaking of the ionic lock that regulates activation. These results provide a comprehensive view of how retinal isomerization triggers helix motion and activation in this prototypical G protein-coupled receptor. © 2009 Nature America, Inc. All rights reserved

The multiple faces of self-assembled lipidic systems

Lipids, the building blocks of cells, common to every living organisms, have the propensity to self-assemble into well-defined structures over short and long-range spatial scales. The driving forces have their roots mainly in the hydrophobic effect and electrostatic interactions. Membranes in lamellar phase are ubiquitous in cellular compartments and can phase-separate upon mixing lipids in different liquid-crystalline states. Hexagonal phases and especially cubic phases can be synthesized and observed in vivo as well. Membrane often closes up into a vesicle whose shape is determined by the interplay of curvature, area difference elasticity and line tension energies, and can adopt the form of a sphere, a tube, a prolate, a starfish and many more. Complexes made of lipids and polyelectrolytes or inorganic materials exhibit a rich diversity of structural morphologies due to additional interactions which become increasingly hard to track without the aid of suitable computer models. From the plasma membrane of archaebacteria to gene delivery, self-assembled lipidic systems have left their mark in cell biology and nanobiotechnology; however, the underlying physics is yet to be fully unraveled