Recent discoveries suggest that a novel second messenger, bis-(335)-cyclic di-GMP (c-diGMP), is extensively used by bacteria to control multicellular behavior. Condensation of two GTP to the dinucleotide is catalyzed by the widely distributed diguanylate cyclase (DGC or GGDEF) domain that occurs in various combinations with sensory and͞or regulatory modules. The crystal structure of the unorthodox response regulator PleD from Caulobacter crescentus, which consists of two CheY-like receiver domains and a DGC domain, has been solved in complex with the product cdiGMP. PleD forms a dimer with the CheY-like domains (the stem) mediating weak monomer-monomer interactions. The fold of the DGC domain is similar to adenylate cyclase, but the nucleotidebinding mode is substantially different. The guanine base is Hbonded to Asn-335 and Asp-344, whereas the ribosyl and ␣-phosphate moieties extend over the ␤2-␤3-hairpin that carries the GGEEF signature motif. In the crystal, c-diGMP molecules are crosslinking active sites of adjacent dimers. It is inferred that, in solution, the two DGC domains of a dimer align in a two-fold symmetric way to catalyze c-diGMP synthesis. Two mutually intercalated c-diGMP molecules are found tightly bound at the stem-DGC interface. This allosteric site explains the observed noncompetitive product inhibition. We propose that product inhibition is due to domain immobilization and sets an upper limit for the concentration of this second messenger in the cell.allosteric regulation ͉ signal transduction ͉ x-ray crystallography

Recent discoveries suggest that a novel second messenger, bis-(335)-cyclic di-GMP (c-diGMP), is extensively used by bacteria to control multicellular behavior. Condensation of two GTP to the dinucleotide is catalyzed by the widely distributed diguanylate cyclase (DGC or GGDEF) domain that occurs in various combinations with sensory and͞or regulatory modules. The crystal structure of the unorthodox response regulator PleD from Caulobacter crescentus, which consists of two CheY-like receiver domains and a DGC domain, has been solved in complex with the product cdiGMP. PleD forms a dimer with the CheY-like domains (the stem) mediating weak monomer-monomer interactions. The fold of the DGC domain is similar to adenylate cyclase, but the nucleotidebinding mode is substantially different. The guanine base is Hbonded to Asn-335 and Asp-344, whereas the ribosyl and ␣-phosphate moieties extend over the ␤2-␤3-hairpin that carries the GGEEF signature motif. In the crystal, c-diGMP molecules are crosslinking active sites of adjacent dimers. It is inferred that, in solution, the two DGC domains of a dimer align in a two-fold symmetric way to catalyze c-diGMP synthesis. Two mutually intercalated c-diGMP molecules are found tightly bound at the stem-DGC interface. This allosteric site explains the observed noncompetitive product inhibition. We propose that product inhibition is due to domain immobilization and sets an upper limit for the concentration of this second messenger in the cell.allosteric regulation ͉ signal transduction ͉ x-ray crystallography

The H + /ATP ratio and the standard Gibbs free energy of ATP synthesis were determined with a new method using a chemiosmotic model system. The puri®ed H + -translocating ATP synthase from chloroplasts was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. During reconstitution, the internal phase was equilibrated with the reconstitution medium, and thereby the pH of the internal liposomal phase, pH in , could be measured with a conventional glass electrode. The rates of ATP synthesis and hydrolysis were measured with the luciferin/luciferase assay after an acid±base transition at different [ATP]/ ([ADP][P i ]) ratios as a function of DpH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the DpH at equilibrium, DpH (eq) (zero net rate), was determined. The analysis of the [ATP]/ ([ADP][P i ]) ratio as a function of DpH (eq) and of the transmembrane electrochemical potential difference, Dm H + (eq), resulted in H + /ATP ratios of 3.9 6 0.2 at pH 8.45 and 4.0 6 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 6 2 kJ/mol at pH 8.45 and 36 6 3 kJ/mol at pH 8.05.

Background:The bacterial second messenger cyclic di-GMP (c-di-GMP) is degraded by EAL phosphodiesterases. Results:The isolated EAL domain is active only as a homodimer. Substrate binding is coupled with EAL dimerization. Conclusion: Activity of many full-length EAL phosphodiesterases may be regulated by catalytic domain dimerization. Significance: A generic mechanism for the regulation of a central node of c-di-GMP signaling is provided.

Photophosphorylation has been reconstituted in a liposomal system containing reaction centers of photosystem I and coupling-factor complex, both highly purified from spinach chloroplasts.This energy-converting model system was put together by diluting the preparation of the couplingfactor complex with an aqueous suspension of proteolipid vesicles, preformed from photosystem