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In bacteria, the monopolar localization of enzymes and protein complexes can result in a bimodal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here, we show that direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the FimV domain of the polar landmark protein HubP is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface-adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations, generated by differences in abundance and timing of polar appearance of PdeB, orchestrates the population behavior with respect to cell-surface interaction and environmental spreading.

Purpose It was shown by several experimental studies that some G protein coupled receptors (GPCR) are sensitive to sodium ions. Furthermore, mutagenesis studies or the determination of crystal structures of the adenosine A2A or δ-opioid receptor revealed an allosteric Na+ binding pocket near to the highly conserved Asp2.50. Within a previous study, the influence of NaCl concentration onto the steady-state GTPase activity at the human histamine H3 receptor (hH3R) in presence of the endogenous histamine or the inverse agonist thioperamide was analyzed. The purpose of the present study was to examine and quantify the Na+-sensitivity of hH3R on a molecular level. Methods To achieve this, we developed a set of equations, describing constitutive activity and the different ligand-receptor equilibria in absence or presence of sodium ions. Furthermore, in order to gain a better understanding of the ligand- and Na+-binding to hH3R on molecular level, we performed molecular dynamic (MD) simulations. Results The analysis of the previously determined experimental steady-state GTPase data with the set of equations presented within this study, reveals that thioperamide binds into the orthosteric binding pocket of the hH3R in absence or presence of a Na+ in its allosteric binding site. However, the data suggest that thioperamide binds preferentially into the hH3R in absence of a sodium ion in its allosteric site. These experimental results were supported by MD simulations of thioperamide in the binding pocket of the inactive hH3R. Furthermore, the MD simulations revealed two different binding modes for thioperamide in presence or absence of a Na+ in its allosteric site. Conclusion The mathematical model presented within this study describes the experimental data regarding the Na+-sensitivity of hH3R in an excellent manner. Although the present study is focused onto the Na+-sensitivity of the hH3R, the resulting equations, describing Na+- and ligand-binding to a GPCR, can be used for all other ion-sensitive GPCRs.

The human histamine H₃ receptor (hH₃R) is a G-protein coupled receptor (GPCR), which modulates the release of various neurotransmitters in the central and peripheral nervous system and therefore is a potential target in the therapy of numerous diseases. Although ligands addressing this receptor are already known, the discovery of alternative lead structures represents an important goal in drug design. The goal of this work was to study the hH3R and its antagonists by means of molecular modelling tools. For this purpose, a strategy was pursued in which a homology model of the hH₃R based on the crystal structure of bovine rhodopsin was generated and refined by molecular dynamics simulations in a dipalmitoylphosphatidylcholine (DPPC)/water membrane mimic before the resulting binding pocket was used for high-throughput docking using the program GOLD. Alternatively, a pharmacophore-based procedure was carried out where the alleged bioactive conformations of three different potent hH₃R antagonists were used as templates for the generation of pharmacophore models. A pharmacophore-based screening was then carried out using the program Catalyst. Based upon a database of 418 validated hH₃R antagonists both strategies could be validated in respect of their performance. Seven hits obtained during this screening procedure were commercially purchased, and experimentally tested in a [³H]Nα-methylhistamine binding assay. The compounds tested showed affinities at hH₃R with Ki values ranging from 0.079 to 6.3 lM.

Imidazolylpropylguanidines derived from impromidine and arpromidine are more potent and efficacious agonists at the guinea pig histamine H₂ receptor (gpH₂R) than at the human H₂R (hH₂R) in the GTPase assay. Additionally, such guanidines are histamine H₁ receptor (H₁R) antagonists with preference for the human relative to the guinea pig receptor. The purpose of this study was to examine structure-activity relationships of guanidines at human and guinea pig H₁R and H₂R species isoforms expressed in Sf9 insect cells. Three impromidine analogues and six arpromidine analogues exhibited agonistic activity at H₂R and antagonistic activity at H₁R as assessed in the steady-state GTPase assay. Species selectivity of derivatives was similar as compared with the parent compounds. None of the structural modifications examined (different aromatic ring systems and different ring substituents) was superior in terms of H₂R potency and efficacy relative to impromidine and arpromidine, respectively. These data point to substantial structural constraints at the agonist binding site of H₂R. Guanidines exhibited distinct structure-activity relationships for H₁R antagonism in a radioligand competition binding assay and the GTPase assay and for H₁R inverse agonism. Our data indicate that it is difficult to obtain guanidine-type agonists with high potency and high efficacy for hH₂R, but those compounds may be useful tools for exploring the antagonist binding site and constitutive activity of H₁R

Amphiphilic substances may stimulate cellular events through direct activation of G-proteins. The present experiments indicate that several amphiphilic sweeteners and the bitter tastant, quinine, activate transducin and Gi/Go-proteins. Concentrations of taste substances required to activate G-proteins in vitro correlated with those used to elicit taste. These data support the hypothesis that amphiphilic taste substances may elicit taste through direct activation of G-proteins.

In plasma membranes of human neutrophils, we identified two major pertussis toxin substrates of 40 kDa Mr with pI values of 5.30 and 5.37. Only the acidic of the two substrates was also present in neutrophil cytosol. Two-dimensional tryptic peptide maps revealed a high degree of homology of cytosolic and particulate substrates. Purified G-protein beta gamma-complex stimulated pertussis toxin-catalyzed [32P]ADP-ribosylation of membranous and cytosolic substrates of neutrophils less than 2-fold and 6-fold, respectively. Hydrodynamic properties of the cytosolic substrate strongly suggested that it exists as a monomer. Purified G-protein beta gamma-complex increased the s20,w value of the cytosolic substrate from 3.3 S to 4.0 S. The GTP analogue, guanosine 5'-O-(3-thiotriphosphate), promoted the release of pertussis toxin substrates from plasma membranes. An antiserum raised against a sequence specific for the Gi2 alpha-subunit reacted with 39-40 kDa proteins in plasma membranes and with an apparently single 40 kDa protein in cytosol. We conclude that neutrophil cytosol contains monomeric Gi2 alpha-subunits which--by interacting with hydrophobic beta gamma-complexes--may reversibly bind to the plasma membrane.

Catechol-O-methyltransferase (COMT) degrades catecholamines, such as dopamine and epinephrine, by methylating them in the presence of a divalent metal cation (usually Mg(II)), and S-adenosyl-L-methionine. The enzymatic activity of COMT is known to be vitally dependent on the nature of the bound metal: replacement of Mg(II) with Ca(II) leads to a complete deactivation of COMT; Fe(II) is slightly less than potent Mg(II), and Fe(III) is again an inhibitor. Considering the fairly modest role that the metal plays in the catalyzed reaction, this dependence is puzzling, and to date remains an enigma. Using a quantum mechanical / molecular mechanical dynamics method for extensive sampling of protein structure, and first principle quantum mechanical calculations for the subsequent mechanistic study, we explicate the effect of metal substitution on the rate determining step in the catalytic cycle of COMT, the methyl transfer. In full accord with experimental data, Mg(II) bound to COMT is the most potent of the studied cations and it is closely followed by Fe(II), whereas Fe(III) is unable to promote catalysis. In the case of Ca(II), a repacking of the protein binding site is observed, leading to a significant increase in the activation barrier and higher energy of reaction. Importantly, the origin of the effect of metal substitution is different for different metals: for Fe(III) it is the electronic effect, whereas in the case of Ca(II) it is instead the effect of suboptimal protein structure.