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Phosphorylation-independent regulation of the diguanylate cyclase WspR.

De N, Pirruccello M, Krasteva PV, Bae N, Raghavan RV, Sondermann H - PLoS Biol. (2008)

Bottom Line: Moreover, our data suggest that these enzymes can be activated by phosphodiesterases.Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling.A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation.

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SEC-Coupled Multiangle Light-Scattering Analysis of Purified WspR in Solution(A) Monomeric BSA (4 mg/ml; Sigma) was analyzed by coupled SEC/multiangle light scattering. The mobile phase consists of 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM DTT. In the left panel, the primary signal (in volts) is plotted against the elution volume. The solid (colored) trace shows the signal of one of the light-scattering detectors (at 90° to the incident beam). The signal from the refractive index detector is shown as a dashed line. The grey area highlights the analyzed peak. The void volume (void) and the end of the experiment (buffer) are indicated. In the right panel, molecular weights, determined by light scattering, and protein concentration, measured by change of refractive index, from each data slice (0.5-s increments) are plotted against the elution volume. The grey horizontal line indicates the theoretical molecular weight.(B–E) Cyclic di-GMP–bound WspRwt (4 mg/ml) (B), PDE-treated WspRwt (4 mg/ml) (C), WspRGGAAF (4 mg/ml) (D), and WspRR242A (4 mg/ml) (E) were analyzed as described in (A). The grey horizontal lines indicate the theoretical molecular weight for monomers, dimers, and tetramers.
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pbio-0060067-g003: SEC-Coupled Multiangle Light-Scattering Analysis of Purified WspR in Solution(A) Monomeric BSA (4 mg/ml; Sigma) was analyzed by coupled SEC/multiangle light scattering. The mobile phase consists of 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM DTT. In the left panel, the primary signal (in volts) is plotted against the elution volume. The solid (colored) trace shows the signal of one of the light-scattering detectors (at 90° to the incident beam). The signal from the refractive index detector is shown as a dashed line. The grey area highlights the analyzed peak. The void volume (void) and the end of the experiment (buffer) are indicated. In the right panel, molecular weights, determined by light scattering, and protein concentration, measured by change of refractive index, from each data slice (0.5-s increments) are plotted against the elution volume. The grey horizontal line indicates the theoretical molecular weight.(B–E) Cyclic di-GMP–bound WspRwt (4 mg/ml) (B), PDE-treated WspRwt (4 mg/ml) (C), WspRGGAAF (4 mg/ml) (D), and WspRR242A (4 mg/ml) (E) were analyzed as described in (A). The grey horizontal lines indicate the theoretical molecular weight for monomers, dimers, and tetramers.

Mentions: The results for mutant and wild-type proteins discussed above are shown in Figure 3 and summarized in Table 1 (including nucleotide binding and activity data). Bovine serum albumin (BSA) served as an isotropically scattering sample for the normalization of the light-scattering detectors (Figure 3A). The averaged molecular weight measured across the peak corresponding to monomeric BSA (70.4 kDa, with a polydispersity index of 1.003) is in good agreement with the theoretical value (66.4 kDa).


Phosphorylation-independent regulation of the diguanylate cyclase WspR.

De N, Pirruccello M, Krasteva PV, Bae N, Raghavan RV, Sondermann H - PLoS Biol. (2008)

SEC-Coupled Multiangle Light-Scattering Analysis of Purified WspR in Solution(A) Monomeric BSA (4 mg/ml; Sigma) was analyzed by coupled SEC/multiangle light scattering. The mobile phase consists of 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM DTT. In the left panel, the primary signal (in volts) is plotted against the elution volume. The solid (colored) trace shows the signal of one of the light-scattering detectors (at 90° to the incident beam). The signal from the refractive index detector is shown as a dashed line. The grey area highlights the analyzed peak. The void volume (void) and the end of the experiment (buffer) are indicated. In the right panel, molecular weights, determined by light scattering, and protein concentration, measured by change of refractive index, from each data slice (0.5-s increments) are plotted against the elution volume. The grey horizontal line indicates the theoretical molecular weight.(B–E) Cyclic di-GMP–bound WspRwt (4 mg/ml) (B), PDE-treated WspRwt (4 mg/ml) (C), WspRGGAAF (4 mg/ml) (D), and WspRR242A (4 mg/ml) (E) were analyzed as described in (A). The grey horizontal lines indicate the theoretical molecular weight for monomers, dimers, and tetramers.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2270323&req=5

pbio-0060067-g003: SEC-Coupled Multiangle Light-Scattering Analysis of Purified WspR in Solution(A) Monomeric BSA (4 mg/ml; Sigma) was analyzed by coupled SEC/multiangle light scattering. The mobile phase consists of 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM DTT. In the left panel, the primary signal (in volts) is plotted against the elution volume. The solid (colored) trace shows the signal of one of the light-scattering detectors (at 90° to the incident beam). The signal from the refractive index detector is shown as a dashed line. The grey area highlights the analyzed peak. The void volume (void) and the end of the experiment (buffer) are indicated. In the right panel, molecular weights, determined by light scattering, and protein concentration, measured by change of refractive index, from each data slice (0.5-s increments) are plotted against the elution volume. The grey horizontal line indicates the theoretical molecular weight.(B–E) Cyclic di-GMP–bound WspRwt (4 mg/ml) (B), PDE-treated WspRwt (4 mg/ml) (C), WspRGGAAF (4 mg/ml) (D), and WspRR242A (4 mg/ml) (E) were analyzed as described in (A). The grey horizontal lines indicate the theoretical molecular weight for monomers, dimers, and tetramers.
Mentions: The results for mutant and wild-type proteins discussed above are shown in Figure 3 and summarized in Table 1 (including nucleotide binding and activity data). Bovine serum albumin (BSA) served as an isotropically scattering sample for the normalization of the light-scattering detectors (Figure 3A). The averaged molecular weight measured across the peak corresponding to monomeric BSA (70.4 kDa, with a polydispersity index of 1.003) is in good agreement with the theoretical value (66.4 kDa).

Bottom Line: Moreover, our data suggest that these enzymes can be activated by phosphodiesterases.Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling.A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation.

Show MeSH
Related in: MedlinePlus