Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization
2007; Elsevier BV; Volume: 282; Issue: 40 Linguagem: Inglês
10.1074/jbc.m704702200
ISSN1083-351X
AutoresRalf Paul, Sören Abel, Paul Wassmann, Andreas Beck, Heiko Heerklotz, Urs Jenal,
Tópico(s)Vibrio bacteria research studies
ResumoDiguanylate 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. 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. Cyclic 3′,5′-guanylyl and adenylyl nucleotides function as second messengers in signal transduction pathways of eukaryotes and prokaryotes. The synthesis of these molecules is catalyzed by a wide variety of nucleotidyl cyclases, which are active as homo- or heterodimers (1Sinha S.C. Sprang S.R. Rev. Physiol. Biochem. Pharmacol. 2006; 157: 105-140Crossref PubMed Scopus (70) Google Scholar). Monocyclic nucleotidyl cyclases that catalyze the formation of cAMP or cGMP are regulated by small molecules, endogenous domains, or exogenous protein partners, many of which alter the interface of the catalytic domains and therefore the integrity of the catalytic site. Much less is known about catalysis and regulation mechanisms of the recently discovered family of diguanylate cyclases (DGCs). 3The abbreviations used are:DGCdiguanylate cyclasec-di-GMPcyclic diguanylic acidSECsize-exclusion chromatographyITCisothermal titration calorimetryDSSdisuccinimidyl suberatePVDFpolyvinylidene difluorideGFPgreen fluorescent protein.3The abbreviations used are:DGCdiguanylate cyclasec-di-GMPcyclic diguanylic acidSECsize-exclusion chromatographyITCisothermal titration calorimetryDSSdisuccinimidyl suberatePVDFpolyvinylidene difluorideGFPgreen fluorescent protein. DGCs are responsible for the synthesis of cyclic di-GMP, a ubiquitous second messenger involved in bacterial bio-film formation and persistence (2Jenal U. Malone J. Annu. Rev. Genet. 2006; 40: 385-407Crossref PubMed Scopus (503) Google Scholar). Cellular levels of c-di-GMP are controlled through the opposing activities of DGCs and phosphodiesterases, which form two large families of output domains found in bacterial one- and two-component systems (3Ulrich L.E. Koonin E.V. Zhulin I.B. Trends Microbiol. 2005; 13: 52-56Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). The DGC activity is contained within the highly conserved GGDEF domain, whose three-dimensional fold is similar to the catalytic core of adenylate cyclase and the “palm” domain of DNA polymerases (4Paul R. Weiser S. Amiot N.C. Chan C. Schirmer T. Giese B. Jenal U. Genes Dev. 2004; 18: 715-727Crossref PubMed Scopus (487) Google Scholar, 5Chan C. Paul R. Samoray D. Amiot N.C. Giese B. Jenal U. Schirmer T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17084-17089Crossref PubMed Scopus (362) Google Scholar). Because GGDEF domains are often associated with sensory input domains, it was proposed that these regulatory proteins serve to directly couple environmental or internal stimuli to a specific cellular response through the synthesis of the second messenger c-di-GMP. Similar to monocyclic nucleotidyl cyclases, the controlled formation of catalytically competent GGDEF domain dimers may be a key mode of DGC regulation (2Jenal U. Malone J. Annu. Rev. Genet. 2006; 40: 385-407Crossref PubMed Scopus (503) Google Scholar, 5Chan C. Paul R. Samoray D. Amiot N.C. Giese B. Jenal U. Schirmer T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17084-17089Crossref PubMed Scopus (362) Google Scholar, 6Ryjenkov D.A. Tarutina M. Moskvin O.V. Gomelsky M. J. Bacteriol. 2005; 187: 1792-1798Crossref PubMed Scopus (462) Google Scholar). A simple model proposes that dimerization mediates an antiparallel arrangement of two DGC domains, each of which is loaded with one GTP substrate molecule. Such an arrangement would allow deprotonation of the GTP 3′OH groups and subsequent intermolecular nucleophilic attacks onto the α-phosphate to occur (5Chan C. Paul R. Samoray D. Amiot N.C. Giese B. Jenal U. Schirmer T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17084-17089Crossref PubMed Scopus (362) Google Scholar). diguanylate cyclase cyclic diguanylic acid size-exclusion chromatography isothermal titration calorimetry disuccinimidyl suberate polyvinylidene difluoride green fluorescent protein. diguanylate cyclase cyclic diguanylic acid size-exclusion chromatography isothermal titration calorimetry disuccinimidyl suberate polyvinylidene difluoride green fluorescent protein. The diguanylate cyclase PleD controls pole morphogenesis during the Caulobacter crescentus cell cycle (4Paul R. Weiser S. Amiot N.C. Chan C. Schirmer T. Giese B. Jenal U. Genes Dev. 2004; 18: 715-727Crossref PubMed Scopus (487) Google Scholar, 7Hecht G.B. Newton A. J. Bacteriol. 1995; 177: 6223-6229Crossref PubMed Google Scholar, 8Aldridge P. Jenal U. Mol. Microbiol. 1999; 32: 379-391Crossref PubMed Scopus (102) Google Scholar, 9Aldridge P. Paul R. Goymer P. Rainey P. Jenal U. Mol. Microbiol. 2003; 47: 1695-1708Crossref PubMed Scopus (190) Google Scholar, 10Levi A. Jenal U. J. Bacteriol. 2006; 188: 5315-5318Crossref PubMed Scopus (58) Google Scholar). PleD is an unorthodox member of the response regulator family of two-component signal transduction systems with two receiver domains arranged in tandem fused to a GGDEF output domain (5Chan C. Paul R. Samoray D. Amiot N.C. Giese B. Jenal U. Schirmer T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17084-17089Crossref PubMed Scopus (362) Google Scholar). Phosphorylation by two cognate kinases, PleC and DivJ, is required for the activation and dynamic sequestration of PleD to the differentiating pole (4Paul R. Weiser S. Amiot N.C. Chan C. Schirmer T. Giese B. Jenal U. Genes Dev. 2004; 18: 715-727Crossref PubMed Scopus (487) Google Scholar, 9Aldridge P. Paul R. Goymer P. Rainey P. Jenal U. Mol. Microbiol. 2003; 47: 1695-1708Crossref PubMed Scopus (190) Google Scholar). Although the first receiver domain (Rec1) serves as phosphoryl acceptor (at the conserved Asp-53 residue), the second receiver domain (Rec2) was proposed to function as an adaptor for dimerization of activated PleD (5Chan C. Paul R. Samoray D. Amiot N.C. Giese B. Jenal U. Schirmer T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17084-17089Crossref PubMed Scopus (362) Google Scholar, 9Aldridge P. Paul R. Goymer P. Rainey P. Jenal U. Mol. Microbiol. 2003; 47: 1695-1708Crossref PubMed Scopus (190) Google Scholar). A simple mechanistic model for the activation of PleD proposes that phosphorylation at the conserved Asp-53 of Rec1 induces repacking of the Rec1/Rec2 interface. This in turn would mediate dimer formation by isologous Rec1-Rec2 contacts across the interface and thereby facilitate reorientation and assembly of two C-terminal DGC domains (5Chan C. Paul R. Samoray D. Amiot N.C. Giese B. Jenal U. Schirmer T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17084-17089Crossref PubMed Scopus (362) Google Scholar). Here we demonstrate that PleD activity can be greatly stimulated in vitro by the phosphoryl mimic BeF3 and that activation of PleD results in dimer formation. Cross-linking experiments revealed that the DGC activity resides entirely in the dimer fraction of activated PleD. Furthermore, controlled dimerization not only modulates DGC activity but is also employed to couple PleD activity to its subcellular sequestration. This is the first demonstration that GGDEF protein dimers represent the active conformation of diguanylate cyclases and confirms that oligomerization can be used to regulate the activity of this abundant class of signaling proteins. Strains, Plasmids, and Media—Bacterial strains and plasmids used in this study are shown in Table 1. Escherichia coli strains were grown in Luria Broth (LB) media supplemented with antibiotics for selection, when necessary. The exact procedure of strain and plasmid construction is available on request.TABLE 1Activities of PleD wild-type and mutant proteinsProteinWithout BeF3With BeF3nanomoles of c-di-GMP min-1 mg-1PleD3.32 (±0.7)aIn parentheses: standard deviation.159.97 (±22.6)PleDD53N2.38 (±0.3)1.10 (±0.2)PleDY26ANDbND, not detectable.0.036 (±0.017)a In parentheses: standard deviation.b ND, not detectable. Open table in a new tab Expression and Purification of PleD—E. coli cells carrying the respective expression plasmid were grown in 200 ml of LB medium with ampicillin (100 μg/ml), and expression was induced by adding isopropyl 1-thio-β-d-galactopyranoside to 0.4 mm final concentration. After harvesting by centrifugation, the cells were resuspended in TN buffer (50 mm Tris-HCl at pH 8.0, 500 mm NaCl, 5 mm β-mercaptoethanol) and lysed by passage through a French press cell. The suspension was clarified by centrifugation, followed by a high spin centrifugation step (100,000 × g, 1 h). The supernatant was loaded onto Ni-NTA affinity resin (Qiagen), washed with TN buffer, and eluted with an imidazole gradient. Elution fractions were examined for purity by SDS-PAGE, and fractions containing pure protein were pooled. PleD was extensively dialyzed first against 500 mm NaCl, 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, pH 8.0, 5 mm β-mercaptoethanol, and than against 250 mm NaCl, 25 mm Tris-HCl, pH 7.8, 5 mm β-mercaptoethanol. Prior to cross-linking experiments PleD was dialyzed against a buffer containing 250 mm NaCl, 5 mm PO4, and 5 mm β-mercaptoethanol. Analytical size exclusion chromatography (SEC) was performed with a Superdex 200 column on a SMART system (Amersham Biosciences) at a flow rate of 50 μl/min. Preparative SEC to quantitatively strip nickel-nitrilotriacetic acid-purified PleD from bound c-di-GMP was performed on a preparative scale Superdex 200 column on an AEKTA system (Amersham Biosciences). Enzymatic Assays—Diguanylate cyclase assays were adapted from procedures described previously (Paul et al. 4). The standard reaction mixtures with purified PleD contained 50 mm Tris-HCl, pH 7.8, 250 mm NaCl, 10 mm MgCl2 in a 50-μl volume and were started by adding 100 μm GTP/[α-33P]GTP (PerkinElmer Life Sciences, 0.01 μCi/μl). To calculate the initial velocity of product formation, aliquots were withdrawn at regular time intervals, and the reaction was stopped with an equal volume of 50 mm EDTA, pH 6.0. Reaction products (2 μl) were separated on polyethyleneimine-cellulose plates (Macherey-Nagel) in 1.5 m KH2PO4/5.5 M (NH4)2SO4 (pH 3.5), mixed in a 2:1 ratio. Plates were exposed to a phosphorimaging screen, and the intensity of the various radioactive species was calculated by quantifying the intensities of the relevant spots using Image-QuaNT software (Amersham Biosciences). Measurements were always restricted to the linear range of product formation. Cross-linking Assays—The purified protein (20 or 25 μm in 100 mm NaCl, 5 mm NaPO4, pH 7.8, 10 mm MgCl2,5 mm β-mercaptoethanol, ± 1 mm BeCl2/10 mm NaF) was incubated with 2 mm disuccinimidyl suberate (DSS, Pierce) for 0, 1, 5, and 10 min. The cross-linker was inactivated by adding Tris-HCl, pH 7.8, to 50 mm final concentration. After separation on 10% SDS-PAGE and transfer to a PVDF membrane, PleD monomeric and dimeric forms were detected by staining with an anti-PleD antibody (8Aldridge P. Jenal U. Mol. Microbiol. 1999; 32: 379-391Crossref PubMed Scopus (102) Google Scholar). Isothermal Titration Calorimetry—The interaction of PleD with cyclic-di-GMP was measured with a VP-ITC isothermal titration calorimeter from MicroCal (Northampton, MA), with 3 μm PleD in the cell and 90 μm c-di-GMP in the syringe (buffer: 100 mm NaCl, 25 mm Tris-HCl, pH 7.8, 5 mm MgCl2, and 1 mm β-mercaptoethanol). All solutions were thoroughly degassed and equilibrated to 25 °C before filling into the calorimeter. The delay between the injections was set to 5–10 min to ensure complete re-equilibration between subsequent injections. The heat capacity of the interaction between the inhibitor and the protein was estimated through measurements between 5 and 25 °C. Microscopy and Photography—C. crescentus strains were grown in 5 ml of peptone-yeast extract media containing 5 μg/ml tetracycline (PYE/tet) for 18 h at 30 °C on a roller incubator. The stationary phase cultures were diluted 1/50 and grown for another 8–10 h in 5 ml of PYE/tet. For fluorescence imaging 1 μl of bacterial culture was placed on a microscope slide layered with a pad of 2% agarose dissolved in water. An Olympus IX71 microscope equipped with an UPlanSApo 100×/1.40 oil objective (Olympus) and a coolSNAP HQ (Photometrics) charge-coupled device camera were used to take differential interference contrast and fluorescence photomicrographs. For GFP fluorescence fluorescein isothiocyanate filter sets (Ex 490/20 nm, Em 528/38 nm) were used with exposure times of 0.15 and 1.0 s, respectively. Images were processed with softWoRx version 3.3.6 and Photoshop CS version 8.0. Activation of the PleD Diguanylate Cyclase by Beryllium Fluoride—To investigate the specific requirements for PleD DGC activity and activation in vitro, we first set out to define the optimal reaction conditions with respect to pH, the concentrations of monovalent and divalent cations, and protein concentration. PleD was enzymatically active between pH 6.5 and 10.0, with maximal activity between pH 7.5 and 8.5 (supplemental Fig. S1). Enzymatic activity was strictly dependent on the presence of either Mg2+ (supplemental Fig. S1) or Mn2+ (not shown), with Mn2+ resulting in a slightly higher activity compared with Mg2+. The strict requirement for bivalent cations is in agreement with the recent finding of coordinating metal ions in the catalytic center of the PleD DGC (11Wassmann P. Chan C. Beck A. Heerklotz H. Paul R. Jenal U. Schirmer T. Structure. 2007; 15: 915-927Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Activity decreased with increasing NaCl concentration (supplemental Fig. S1). Also, the addition of KCl (25 mm) to reaction assays (4Paul R. Weiser S. Amiot N.C. Chan C. Schirmer T. Giese B. Jenal U. Genes Dev. 2004; 18: 715-727Crossref PubMed Scopus (487) Google Scholar) slightly decreased the enzymatic activity and was omitted in subsequent experiments. Activation of the PleD DGC in vitro and in vivo requires the transfer of a phosphoryl group onto the aspartic acid acceptor residue Asp-53 of the first receiver domain (Fig. 1) (4Paul R. Weiser S. Amiot N.C. Chan C. Schirmer T. Giese B. Jenal U. Genes Dev. 2004; 18: 715-727Crossref PubMed Scopus (487) Google Scholar). However, in vitro phosphorylation experiments with PleD resulted in an exiguous increase of DGC activity, possibly due to suboptimal assay conditions or to low stability of the phosphorylated form (4Paul R. Weiser S. Amiot N.C. Chan C. Schirmer T. Giese B. Jenal U. Genes Dev. 2004; 18: 715-727Crossref PubMed Scopus (487) Google Scholar). For this reason we tested activation of PleD by beryllium fluoride (BeF3) a molecular mimic of a phosphoryl group that has been widely used for biochemical and structural studies of bacterial response regulators (12Yan D. Cho H.S. Hastings C.A. Igo M.M. Lee S.Y. Pelton J.G. Stewart V. Wemmer D.E. Kustu S. Proc. Natl. Acad. Sci. U. S. 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As shown in Fig. 2, BeF3 significantly stimulated the enzymatic activity of PleD, with optimal concentrations of 1 mm BeCl2 (Fig. 2A) and 10 mm NaF (Fig. 2B), respectively. DGC activation was reversible and was immediately abolished upon removal of BeF3 (data not shown). A PleD mutant protein lacking the phosphoryl acceptor site Asp-53 (PleDD53N) could not be activated, suggesting that BeF3 activates the protein by specifically interacting with this residue in the first receiver domain (Fig. 2C). A constitutively active mutant of PleD, PleD* (4Paul R. Weiser S. Amiot N.C. Chan C. Schirmer T. Giese B. Jenal U. Genes Dev. 2004; 18: 715-727Crossref PubMed Scopus (487) Google Scholar) was also stimulated by BeF3, but only by a factor of 2 (data not shown). This is consistent with the view that PleD* is locked in an active state (see below). Concentrations of BeCl2 or NaF above 1 mm and 10 mm, respectively, had a negative effect on DGC activity (Fig. 2, A–C). At these concentrations BeF3 probably interacts nonspecifically with surface residues that are required for diguanylate cyclase activity.FIGURE 2Beryllium fluoride-mediated activation of the PleD diaguanylate cyclase. A, PleD (5 μm) activity (nanomoles of c-di-GMP min–1 mg–1) as a function of BeCl2 concentration in the presence of 5 mm NaF. B, PleD (2.5 μm) activity (nanomoles of c-di-GMP min–1 mg–1) as a function of NaF concentration in the presence of 1 mm BeCl2. C, PleDD53N (5 μm) activity (nanomoles of c-di-GMP min–1 mg–1) as a function of BeCl2 concentration in the presence of 5 mm NaF. D, specific activities (nanomoles of c-di-GMP min–1 mg–1) for PleD (open circles), PleD activated with BeF3 (filled circles), and the constitutively active PleD* mutant protein (triangles) as a function of the protein concentration.View Large Image Figure ViewerDownload Hi-res image Download (PPT) BeF3 Activation Results in PleD Dimerization—The specific activities of non-activated and BeF3-activated PleD wild-type protein and of the constitutively active PleD* mutant increased with increasing protein concentration (Fig. 2D). This suggested that PleD might be active in a dimeric (or oligomeric) form, with dimer formation being concentration-dependent. The observation that PleD* and BeF3 activated PleD reach an activity plateau at much lower protein concentrations than non-activated PleD further suggested that the PleD dimerization constant is affected either by genetic changes or by chemical activation of the protein. To test this hypothesis, cross-linking studies were performed with PleD using the chemical cross-linker DSS (see “Materials and Methods”). We reasoned that the amount of covalently cross-linked dimers was proportional to the amount of dimers in solution. When the DSS cross-linker was incubated with non-activated wild-type PleD or PleDD53N at a protein concentration of 20 μm (well below the estimated dissociation constant Kd of dimerization of 104 μm (11Wassmann P. Chan C. Beck A. Heerklotz H. Paul R. Jenal U. Schirmer T. Structure. 2007; 15: 915-927Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar)), only a minor fraction of the protein was captured as covalently cross-linked dimers (Fig. 3A). This is consistent with the low basal level of enzymatic activity observed for non-activated PleD (Table 1). Activation of PleD by BeF3 not only increased DGC activity (Table 1) but also the amount of cross-linked dimer species (Fig. 3A). In contrast, the non-activable PleDD53N (Fig. 2 and Table 1) showed no increase in cross-linked dimers (Fig. 3A). The crystal structure of non-activated PleD predicted a specific dimerization interface in the Rec1-Rec2 receiver domain stem with a small contact patch around the surface exposed Tyr residue at position 26 of the first receiver domain (Fig. 1). Tyr-26 is strictly conserved in PleD homologs that share a Rec1-Rec2-DGC domain structure, but not in response regulators with a different domain architecture (supplemental Fig. S2). To test if this residue plays a role in PleD dimerization, DGC activity and dimerization behavior of the PleDY26A mutant protein were analyzed. Indeed, PleDY26A was completely inactive in the absence and only marginally active in the presence of BeF3 (Table 1). Consistent with this, only a minor fraction of the protein could be cross-linked in the dimer form, irrespectively of the presence of BeF3 (Fig. 3A). In agreement with these in vitro data, the pleDY26A allele failed to complement the pleiotropic developmental defects of a C. crescentus pleD null mutant. Together these results strongly support the view that Tyr-26 residue forms part of the interaction surface of PleD dimers. This is consistent with the finding that, although additional inter-chain contacts are formed in the crystal structure of BeF3 activated PleD, the specific contact around Tyr-26 is maintained (11Wassmann P. Chan C. Beck A. Heerklotz H. Paul R. Jenal U. Schirmer T. Structure. 2007; 15: 915-927Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The Diguanylate Cyclase Activity of PleD Resides in the Dimer Fraction—As indicated by cross-linking, PleD forms dimers in the presence of BeF3. To investigate if the enzymatic activity coincided with the cross-linked PleD fractions, reactions were diluted 10-fold immediately after DSS treatment and quenching. At this BeF3 concentration PleD showed only residual DGC activity (Fig. 2). As shown in Fig. 3B, cross-linking of BeF3-activated PleD resulted in a 6-fold increase of DGC activity as compared with non-cross-linked samples. In contrast, cross-linking of PleD in the absence of BeF3 and cross-linking of PleDD53N either in the presence or absence of BeF3 did not result in increased DGC activity. Taken together, these results suggested that, in the presence of BeF3, a fraction of the PleD protein is trapped by DSS cross-link in a dimerized form and that the dimer represents the active conformation of PleD. To further substantiate this idea we attempted to separate active dimers from inactive monomers by SEC (see “Materials and Methods”). Only PleD, but not PleDD53N, changed its apparent molecular size in the presence of BeF3 (Fig. 4, A and B). However, in contrast to the constitutive active PleD*, which is present predominantly as a dimer (Fig. 4C), BeF3-activated PleD did not show resolved monomer and dimer peaks. Rather, the broadening of the PleD peak and its shift to a higher apparent molecular mass are consistent with a rapid equilibrium between monomers and dimers for the activated PleD. Only when BeF3-activated PleD was cross-linked with DSS before SEC, defined monomer and dimer peaks were observed (Fig. 4D). The peak corresponding to cross-linked PleD dimers was considerably smaller than the monomer peak indicating that only a minor fraction of PleD was trapped in the dimeric form. This was confirmed by immunoblot experiments with anti-PleD antibodies that also showed an increase of the dimer form (fractions 11 and 12) in BeF3-treated samples (Fig. 4F). Nevertheless, the dimer fractions almost exclusively accounted for the detectable DGC activity (Fig. 4E). The observations that BeF3 treatment increased the proportion of PleD dimers and that isolated dimers exhibited high DGC activity are in support of an “activation by dimerization” mechanism. DGC Activity Is Not Required for PleD Dimerization—To demonstrate that dimerization is required, but not sufficient for diguanylate cyclase activity, we analyzed if the PleDE370Q mutant formed dimers. The conserved Glu-370 residue is part of the A-site signature sequence GG(E/D)EF of the PleD diguanylate cyclase domain and was proposed to coordinate a Mg2+ ion in the active site required for deprotonation of the 3′-OH group of one GTP substrate molecule and its subsequent nucleophilic attack onto the α-phosphate of the other GTP molecule. As predicted, the PleDE370Q mutant lacked detectable enzymatic activity both in the presence and absence of BeF3 (data not shown). We even failed to detect any enzymatic activity when the protein concentration was increased to >50 μm and with prolonged incubation times. However, in contrast to PleDY26A the lack of activity was not due to a failure to dimerize. When BeF3-activated PleDE370Q was used in cross-link experiments, the behavior of the mutant form was indistinguishable from PleD wild type (supplemental Fig. S3). Thus, dimerization is clearly a prerequisite for, rather than a consequence of diguanylate cyclase activity. Activation of the PleD Diguanylate Cyclase Does Not Interfere with Feedback Inhibition—The diguanylate cyclase activity of PleD is subject to strong product inhibition through binding of c-di-GMP to an allosteric I-site widely conserved among GGDEF domain proteins (5Chan C. Paul R. Samoray D. Amiot N.C. Giese B. Jenal U. Schirmer T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17084-17089Crossref PubMed Scopus (362) Google Scholar, 19Christen B. Christen M. Paul R. Schmid F. Folcher M. Jenoe P. Meuwly M. Jenal U. J. Biol. Chem. 2006; 281: 32015-32024Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). To test if PleD activation with BeF3 interferes with allosteric control, binding of c-di-GMP to the I-site was directly measured using ITC (Fig. 5). Integration of the titration peaks of c-di-GMP injected from the syringe into the cell of the calorimeter containing PleD produced a sigmoidal enthalpy curve for the interaction between PleD and c-di-GMP. The slope of the binding curve implies a dissociation constant of 0.3 μm (±0.1 μm). This is in good agreement with the Ki of 0.5 μm determined earlier (5Chan C. Paul R. Samoray D. Amiot N.C. Giese B. Jenal U. Schirmer T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17084-17089Crossref PubMed Scopus (362) Google Scholar, 19Christen B. Christen M. Paul R. Schmid F. Folcher M. Jenoe P. Meuwly M. Jenal U. J. Biol. Chem. 2006; 281: 32015-32024Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In support of a c-di-GMP dimer bound at each I-site (5Chan C. Paul R. Samoray D. Amiot N.C. Giese B. Jenal U. Schirmer T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17084-17089Crossref PubMed Scopus (362) Google Scholar) the binding stoichiometry was measured as 2.1:1 (±0.2) (c-di-GMP:PleD) (Fig. 5). When binding of c-di-GMP to PleD was compared in the non-activated and BeF3-activated conformation, both binding affinity (0.4 ± 0.1 μm) and stoichiometry (2.1:1 ± 0.2) did not change significantly upon activation. In agreement with this, the IC50 values for PleD inhibition measured at a protein concentration of 5 μm were very similar for the non-activated (5.1 ± 1.4 μm) and the BeF3-activated (5.9 ± 1.3 μm) PleD. From these data we conclude that activation of PleD by dimerization does not interf
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