Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger cyclic di-GMP.

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg(2+), cdG·Ca(2+) complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.

H+/ATP ratio of proton transport-coupled ATP synthesis and hydrolysis catalysed by CF0F1-liposomes.

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 purified 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 deltapH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the deltapH at equilibrium, deltapH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][P(i)]) ratio as a function of deltapH (eq) and of the transmembrane electrochemical potential difference, delta micro approximately (H)(+) (eq), resulted in H(+)/ATP ratios of 3.9 +/- 0.2 at pH 8.45 and 4.0 +/- 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 +/- 2 kJ/mol at pH 8.45 and 36 +/- 3 kJ/mol at pH 8.05.

Structural basis of activity and allosteric control of diguanylate cyclase.

Recent discoveries suggest that a novel second messenger, bis-(3'-->5')-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 and/or 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 c-diGMP. 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 nucleotide-binding mode is substantially different. The guanine base is H-bonded to Asn-335 and Asp-344, whereas the ribosyl and alpha-phosphate moieties extend over the beta2-beta3-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.