Phagocytic activity of marine and freshwater bivalves: in vitro exposure of hemocytes to metals (Ag, Cd, Hg and Zn).

We measured non-specific immune function of various bivalves from marine (Cyrtodaria siliqua, Mactromeris polynyma, Mesosdesma arctatum, Mya arenaria, Mya truncata, Mytilus edulis, Serripes groenlandicus, Siliqua costata) and freshwater environments (Dreissena polymorpha and Elliptio complanata). We used flow cytometry to quantify the phagocytosis of fluorescent microspheres by hemocytes exposed in vitro to increasing levels of various metal compounds (AgNO(3), CdCl(2), CH(3)HgCl, HgCl(2) and ZnCl(2)). In some species, low doses of mercury (organic and inorganic) and Zn suggest a hormesis-like stimulation of phagocytic activity. At higher levels of exposure, all metals tested induced a significant dose-related inhibition of hemocyte phagocytosis. The species-specific sensitivity of the assay was determined by comparing the in vitro exposure using the metal concentration inducing a 50% suppression (EC(50)) of the phagocytic activity. Different species expressed different levels of sensitivity. Our results show the variability of the toxic response of different species within a group of similar organisms. It also highlights the need to consider species-species differences in ecotoxicological risk assessment.

Two-component signal transduction.

Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regulator protein. The histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein. Phosphorylation induces a conformational change in the regulatory domain that results in activation of an associated domain that effects the response. The basic scheme is highly adaptable, and numerous variations have provided optimization within specific signaling systems. The domains of two-component proteins are modular and can be integrated into proteins and pathways in a variety of ways, but the core structures and activities are maintained. Thus detailed analyses of a relatively small number of representative proteins provide a foundation for understanding this large family of signaling proteins.

Evidence for phosphorylation-dependent conformational changes in methylesterase CheB.

Enhancement of methylesterase activity of the response regulator CheB is dependent upon phosphorylation of the N-terminal regulatory domain of the enzyme. This domain plays a dual role in the regulation of methylesterase activity with an inhibitory effect in the unphosphorylated state and a stimulatory effect in the phosphorylated state. Structural studies of the unphosphorylated state have indicated that the basis for the regulatory domain's inhibitory effect is partial blockage of access of substrate to the active site suggesting that the activation upon phosphorylation involves a repositioning of the two domains with respect to each other. We report in this study evidence for phosphorylation-dependent conformational changes in CheB. Differences in rates of proteolytic cleavage by trypsin between the phosphorylated and unphosphorylated states have been observed at three sites in the protein with one site, 113, within the regulatory domain and two sites, 134 and 148, lying within the interdomain linker. These results support the hypothesis for the mechanism for the activation of CheB wherein phosphorylation of a specific aspartate residue within the N-terminal domain results in a propagated conformational change within the regulatory domain leading to a repositioning of its two domains. Presumably, structural changes in the regulatory domain of CheB facilitate a repositioning of the N- and C-terminal domains, leading to stimulation of methylesterase activity.

Stabilization of the phospho-aspartyl residue in a two-component signal transduction system in Thermotoga maritima.

The central signaling pathway in many bacterial regulatory systems involves phosphotransfer between two conserved proteins, a histidine protein kinase, and a response regulator. The occurrence of two-component signaling systems in thermophilic bacteria raises questions of how both the proteins and the labile acyl phosphate of the response regulator are adapted to function at elevated temperatures. Thermotoga maritima HpkA is a transmembrane histidine kinase, and DrrA is its cognate response regulator. Both DrrA and the cytoplasmic region of HpkA (HpkA57) have been expressed in Escherichia coli, purified, and characterized. HpkA57 and DrrA have apparent Tm's of 75 and 90 degreesC, respectively. HpkA57 exhibits ATP-dependent autophosphorylation activity similar to that of histidine kinases from mesophiles, with maximum activity at 70 degreesC. DrrA catalyzes transfer of phosphoryl groups from HpkA57 and exhibits Mg2+-dependent autophosphatase activity, with maximum activity at approximately 80 degreesC. At this temperature, the half-life for phospho-DrrA is approximately 3 min. In the absence of Mg2+, the half-life is 26 min, significantly greater than the half-life of a typical acyl phosphate at 80 degreesC. In the absence of Mg2+, at all temperatures examined, phospho-DrrA exhibits much greater stability than acetyl phosphate. This suggests that the active site of this hyperthermophilic response regulator is designed to protect the phospho-aspartyl residue from hydrolysis.

Activation of methylesterase CheB: evidence of a dual role for the regulatory domain.

The response regulator CheB functions within the bacterial chemotaxis system together with the methyltransferase CheR to control the level of chemoreceptor methylation, influencing the signaling activities of the receptors. CheB catalyzes demethylation of specific methylglutamate residues introduced into the chemoreceptors by CheR. CheB has a two-domain architecture consisting of an N-terminal regulatory domain joined by a linker to a C-terminal effector domain. In the unphosphorylated state of the response regulator, the regulatory domain inhibits the methylesterase activity of the effector domain. Upon phosphorylation of a specific aspartate residue within the regulatory domain, the C-terminal methylesterase activity is stimulated, resulting in the subsequent demethylation of the chemoreceptors. We have investigated the mechanism of regulation of CheB activity by the N-terminal regulatory domain. First, we have found that phosphorylation of the N-terminal domain not only relieves inhibition of the C-terminal methylesterase activity but also provides an enhancement of this activity above that seen for the C-terminal effector domain alone. Second, we have identified mutations in CheB that show an enhancement of methylesterase activity in the absence of phosphorylation. Most of these single-site mutations are localized in the linker region joining the regulatory and effector domains. On the basis of these observations, we propose a model for activation of CheB in which phosphorylation of the regulatory domain results in a reorganization of the domain interface, allowing exposure of the active site to the receptor substrate and simultaneously stimulating methylesterase activity.

Structural basis for methylesterase CheB regulation by a phosphorylation-activated domain.

We report the x-ray crystal structure of the methylesterase CheB, a phosphorylation-activated response regulator involved in reversible modification of bacterial chemotaxis receptors. Methylesterase CheB and methyltransferase CheR modulate signaling output of the chemotaxis receptors by controlling the level of receptor methylation. The structure of CheB, which consists of an N-terminal regulatory domain and a C-terminal catalytic domain joined by a linker, was solved by molecular replacement methods using independent search models for the two domains. In unphosphorylated CheB, the N-terminal domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site. We propose that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site. Structural similarity between the two companion receptor modification enzymes, CheB and CheR, suggests an evolutionary and/or functional relationship. Specifically, the phosphorylated N-terminal domain of CheB may facilitate interaction with the receptors, similar to the postulated role of the N-terminal domain of CheR. Examination of surfaces in the N-terminal regulatory domain of CheB suggests that despite a common fold throughout the response regulator family, surfaces used for protein-protein interactions differ significantly. Comparison between CheB and other response regulators indicates that analogous surfaces are used for different functions and conversely, similar functions are mediated by different molecular surfaces.

Signal transduction in bacteria: molecular mechanisms of stimulus-response coupling.

In bacteria, adaptive responses to changing environmental conditions are mediated by signal transduction systems that involve modular protein domains. Despite great diversity in the integration of domains into different systems, studies of individual components have revealed molecular strategies that are widely applicable. Studies of receptors have advanced our understanding of how information is transmitted across membranes, the determination of three-dimensional structures of domains of histidine protein kinase domains and response regulator proteins has begun to reveal the molecular basis of signaling via two-component phosphoryltransfer pathways, and the description of 'eukaryotic-like' protein domains involved in bacterial signaling has emphasized the universality of intracellular signaling mechanisms.

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)