Studying GGDEF Domain in the Act: Minimize Conformational Frustration to Prevent Artefacts.

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies.

Insights into the GTP-dependent allosteric control of c-di-GMP hydrolysis from the crystal structure of PA0575 protein from Pseudomonas aeruginosa.

Bis-(3'-5')-cyclic diguanylic acid (c-di-GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c-di-GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c-di-GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c-di-GMP-dependent response. Both the c-di-GMP molecule and the proteins interacting with this second messenger are characterized by an extraordinary structural plasticity, which has to be taken into account when defining and possibly predicting c-di-GMP-related processes. Here, we report a structure-function relationship study on the catalytic portion of the PA0575 protein from Pseudomonas aeruginosa, bearing both putative DGC and PDE domains. The kinetic and structural studies indicate that the GGDEF-EAL portion is a GTP-dependent PDE. Moreover, the crystal structure confirms the high degree of conformational flexibility of this module. We combined structural analysis and protein engineering studies to propose the possible molecular mechanism guiding the nucleotide-dependent allosteric control of catalysis; we propose that the role exerted by GTP via the GGDEF domain is to allow the two EAL domains to form a dimer, the species competent to enter PDE catalysis.

Beyond nitrogen metabolism: nitric oxide, cyclic-di-GMP and bacterial biofilms.

The nitrogen cycle pathways are responsible for the circulation of inorganic and organic N-containing molecules in nature. Among these pathways, those involving amino acids, N-oxides and in particular nitric oxide (NO) play strategic roles in the metabolism of microorganisms in natural environments and in host-pathogen interactions. Beyond their role in the N-cycle, amino acids and NO are also signalling molecules able to influence group behaviour in microorganisms and cell-cell communication in multicellular organisms, including humans. In this minireview, we summarise the role of these compounds in the homeostasis of the bacterial communities called biofilms, commonly found in environmental, industrial and medical settings. Biofilms are difficult to eradicate since they are highly resistant to antimicrobials and to the host immune system. We highlight the effect of amino acids such as glutamate, glutamine and arginine and of NO on the signalling pathways involved in the metabolism of 3',5'-cyclic diguanylic acid (c-di-GMP), a master regulator of motility, attachment and group behaviour in bacteria. The study of the metabolic routes involving these N-containing compounds represents an attractive topic to identify targets for biofilm control in both natural and medical settings.