Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation

Abstract

C-di-GMP (3',5' -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity.

Figure 1. Motility/biofilm assay on the PA2771 deletion strain and the activity measurement of Dcsbis.

(a) Swimming motility of WT PAO1 and PA2771 deletion strains (ΔPA2771). (b) Swarming motility of WT PAO1 and ΔPA2771 strains. (c) Biofilm assay of WT PAO1 and ΔPA2771 strains. (d) The DGC activity of full-length Dcsbis, Dcsbis-GGDEF, WspR, and SadC. (e) The DGC activity of full-length Dcsbis in the presence of 5 μM c-di-GMP.

Figure 2. The structure of full-length Dcsbis.

(a) The tightly associated homodimer of Dcsbis. The A-site and I-site of Dcsbis are colored in blue and red, respectively. (b) The overall structure of the Dcsbis monomer. The N-terminal GAF domain is colored in red, while the C-terminal GGDEF domain is colored in yellow. (c,d) The hydrogen bonds and hydrophobic interactions contributing to homodimerization.

Figure 3. The structure of the GGDEF domain of Dcsbis in complex with c-di-GMP and the inhibition loop.

(a) The dimer of the Dcsbis-GGDEF/c-di-GMP complex. The I-site and A-site of Dcsbis are in red and blue, respectively. (b) The superposition of the full-length Dcsbis monomer onto the Dcsbis-GGDEF/c-di-GMP complex dimer. The full-length protein is in lighter colors. (c) The peptide loop (protecting loop, residues 120–125) extending from the GAF domain blocks the inhibition site of Dcsbis. The protecting loop is colored in purple, whereas the I-site is red and the active site is blue. (d) The superposition of GGDEF/c-di-GMP complex onto the full-length structure of Dcsbis to model the position of c-di-GMP into the full-length Dcsbis structure. GGDEF/c-di-GMP are highlighted in gray. A close view of the c-di-GMP modeled into the Dcsbis structure.

Figure 4. The putative ligand-binding pocket of the Dcsbis GAF domain.

(a,b) Surface analysis of Dcsbis. The Dcsbis-GAF cartoon is colored in rainbow. (c) The stereo view of the residues that contribute to the formation of the putative ligand-binding pocket.

Figure 5. Double reciprocal plots of the kinetic study of Dcsbis protein in absent and present of c-di-GMP.

This analysis was done by measuring three independent sets of experiments with 5 substrate concentrations (128 μM, 150 μM, 190 μM, 333 μM, 512 μM). The plots are generated by plotting 1/V as a function 1/[S]. The intercept on the vertical axis is 1/V_max, and the intercept on the horizontal axis is −1/K_M.

Figure 6. The proposed model for the activation and inhibition of Dcsbis.

The model demonstrates the potential regulation mechanism of Dcsbis activity. In the absence of the regulatory ligand, substrate GTP, and product c-di-GMP, the Dcisbis protein dimerizes via the GAF domain, with the I-site blocked by the protecting loop (represented by the full-length Dcsbis structure), and thus is highly active; Upon binding to GTP, the active sites of Dcsbis dimer get close to each other and catalyze the formation of c-di-GMP; c-di-GMP induces slight product inhibition. Binding of the potential regulatory ligand to the GAF domain may induce conformational change in the GAF domain, and thus break the GAF dimerization and lead to the release of the blockage of the I-site. At this condition, the product c-di-GMP could bind to the I-sites of GGDEF domain and bridge the formation of a new dimer (represented by the structure of GGDEF domain/c-di-GMP complex).