Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization

Abstract

Diguanylate cyclases (DGCs) are key enzymes of second messenger signaling in bacteria. Their activity is responsible for the condensation of two GTP molecules into the signaling compound cyclic di-GMP. Despite their importance and abundance in bacteria, catalytic and regulatory mechanisms of this class of enzymes are poorly understood. In particular, it is not clear if oligomerization is required for catalysis and if it represents a level for activity control. To address this question we perform in vitro and in vivo analysis of the Caulobacter crescentus diguanylate cyclase PleD. PleD is a member of the response regulator family with two N-terminal receiver domains and a C-terminal diguanylate cyclase output domain. PleD is activated by phosphorylation but the structural changes inflicted upon activation of PleD are unknown. We show that PleD can be specifically activated by beryllium fluoride in vitro, resulting in dimerization and c-di-GMP synthesis. Cross-linking and fractionation experiments demonstrated that the DGC activity of PleD is contained entirely within the dimer fraction, confirming that the dimer represents the enzymatically active state of PleD. In contrast to the catalytic activity, allosteric feedback regulation of PleD is not affected by the activation status of the protein, indicating that activation by dimerization and product inhibition represent independent layers of DGC control. Finally, we present evidence that dimerization also serves to sequester activated PleD to the differentiating Caulobacter cell pole, implicating protein oligomerization in spatial control and providing a molecular explanation for the coupling of PleD activation and subcellular localization.

Type IV Pilus Assembly in Pseudomonas aeruginosa over a Broad Range of Cyclic di-GMP Concentrations

“…4B), whereas this was not the case for PA0169 overexpressing ⌬fimX bacteria. We also tested whether the expression of an unrelated DGC could suppress the PA103⌬fimX T4P assembly defect, choosing the well-characterized Caulobacter crescentus DGC PleD for this experiment (38). Expression of PleD*, a constitutively active allele of this DGC, restored surface pilin assembly means Ϯ standard deviations (SD) (n ϭ 4) from a representative experiment are shown.…”

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…The apparent similarity of DGCs to type III nucleotide cyclases, as well as the dinucleotide nature of c-di-GMP, implied that GGDEF domains function as homodimers, where two monomers come together to form an active site at the dimer interface (112). Each GGDEF monomer contributes a GTP substrate to the formation of an intermolecular phosphoester bond to another molecule of GTP.…”

The Phosphodiesterase DipA (PA5017) Is Essential for Pseudomonas aeruginosa Biofilm Dispersion

“…The current paradigm in the field is that high concentrations of this molecule correlate with a sessile lifestyle (e.g., biofilm formation), while its absence favors motility (e.g., twitching and swarming) and the free-swimming lifestyle (12). Overall, c-di-GMP plays an important role in biofilm formation by regulating the production of the exopolysaccharide (EPS) matrix, and by controlling motility (in particular swarming), autoaggregation, and adhesiveness (16,21,22,23,28,31,35,36,41,42,48,52,54). Levels of c-di-GMP are enzymatically modulated by diguanylate cyclases (DCG), proteins containing a GGDEF domain (36), and phosphodiesterases (PDE) containing either an EAL domain (51) or an HD-GYP domain (43).…”