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Structure of a diguanylate cyclase from Thermotoga maritima: insights into activation, feedback inhibition and thermostability.

Deepthi A, Liew CW, Liang ZX, Swaminathan K, Lescar J - PLoS ONE (2014)

Bottom Line: Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF domain) from Thermotoga maritima, enables the robust enzymatic production of large quantities of c-di-GMP.To understand the structural correlates of tDGC thermostability, its catalytic mechanism and feedback inhibition, we determined structures of an active-like dimeric conformation with both active (A) sites facing each other and of an inactive dimeric conformation, locked by c-di-GMP bound at the inhibitory (I) site.We also report the structure of a single mutant of tDGC, with the R158A mutation at the I-site, abolishing product inhibition and unproductive dimerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.

ABSTRACT
Large-scale production of bis-3'-5'-cyclic-di-GMP (c-di-GMP) would facilitate biological studies of numerous bacterial signaling pathways and phenotypes controlled by this second messenger molecule, such as virulence and biofilm formation. C-di-GMP constitutes also a potentially interesting molecule as a vaccine adjuvant. Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF domain) from Thermotoga maritima, enables the robust enzymatic production of large quantities of c-di-GMP. To understand the structural correlates of tDGC thermostability, its catalytic mechanism and feedback inhibition, we determined structures of an active-like dimeric conformation with both active (A) sites facing each other and of an inactive dimeric conformation, locked by c-di-GMP bound at the inhibitory (I) site. We also report the structure of a single mutant of tDGC, with the R158A mutation at the I-site, abolishing product inhibition and unproductive dimerization. A comparison with structurally characterized DGC homologues from mesophiles reveals the presence of a higher number of salt bridges in the hyperthermophile enzyme tDGC. Denaturation experiments of mutants disrupting in turn each of the salt bridges unique to tDGC identified three salt-bridges critical to confer thermostability.

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The inactive tDGC dimer (a) The two monomers present in the asymmetric unit are colored in grey and yellow respectively with the two bridging c-di-GMP molecules shown as sticks.The views are along (left) and perpendicular (right) to the non-crystallographic dyad. (b) Stereoview of the dimerization mediated by two c-di-GMP molecules (labeled c-di-GMP1 and c-di-GMP2), bound at the I-site of tDGC, forcing the GGDEF domain in an inhibited conformation, with both A-sites facing away from each other. An omit map (blue mesh) with Fourier coefficients 2Fo-Fc, where the c-di-GMP ligand was omitted from phase calculation is shown at 1σ contour level. Residues from the RxxD motif at the I-site forming hydrogen bonds with the bound c-di-GMP molecules are displayed as sticks and the distances between interacting atoms are displayed. The same color code as in panel a is used.
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pone-0110912-g002: The inactive tDGC dimer (a) The two monomers present in the asymmetric unit are colored in grey and yellow respectively with the two bridging c-di-GMP molecules shown as sticks.The views are along (left) and perpendicular (right) to the non-crystallographic dyad. (b) Stereoview of the dimerization mediated by two c-di-GMP molecules (labeled c-di-GMP1 and c-di-GMP2), bound at the I-site of tDGC, forcing the GGDEF domain in an inhibited conformation, with both A-sites facing away from each other. An omit map (blue mesh) with Fourier coefficients 2Fo-Fc, where the c-di-GMP ligand was omitted from phase calculation is shown at 1σ contour level. Residues from the RxxD motif at the I-site forming hydrogen bonds with the bound c-di-GMP molecules are displayed as sticks and the distances between interacting atoms are displayed. The same color code as in panel a is used.

Mentions: The wild type tDGC protein co-purifies with c-di-GMP and co-crystallizes without any extraneous addition of ligand. The structure was determined at a resolution of 2.27 Å (Tables 1and2). In this crystal form, tDGC adopts a dimeric conformation, where the two monomers are related by a non-crystallographic dyad (Fig. 2). The buried accessible surface area between the two monomers is 1,235 Å2 and their interface is stabilized by one salt bridge and thirteen hydrogen bonds. I-site residues 158-RxxD-161 from the two monomers interact with two molecules of c-di-GMP that are mutually intercalated (Fig. 2a). This symmetrical arrangement is similar to the PleD structure [7], with the four guanyl bases of the two c-di-GMP molecules bound to the primary inhibitory sites of one molecule comprising the 158-RxxD-161 motif and a secondary inhibitory site R115 contributed by the second monomer. Residue R158 plays a key role in the interaction with both c-di-GMP molecules, via its guanidinium moiety that completes the set of stacking interactions (Fig. 2b). In this arrangement, the two monomers are locked in an inactive orientation with their A sites facing away from each other in a catalytically unproductive mode (Fig. 2a). The crystallographic dimer observed in this crystal form is consistent with gel filtration analysis showing that tDGC forms a dimer in solution (Fig. 3a). To demonstrate that dimerization is mediated by c-di-GMP, which co-purifies with tDGC, we incubated the protein with RocR, a PDE from Pseudomonas aeruginosa, to remove the bound c-di-GMP. Remarkably, following enzymatic treatment with RocR, the wild type tDGC protein elutes as a monomer (Fig. 3b).


Structure of a diguanylate cyclase from Thermotoga maritima: insights into activation, feedback inhibition and thermostability.

Deepthi A, Liew CW, Liang ZX, Swaminathan K, Lescar J - PLoS ONE (2014)

The inactive tDGC dimer (a) The two monomers present in the asymmetric unit are colored in grey and yellow respectively with the two bridging c-di-GMP molecules shown as sticks.The views are along (left) and perpendicular (right) to the non-crystallographic dyad. (b) Stereoview of the dimerization mediated by two c-di-GMP molecules (labeled c-di-GMP1 and c-di-GMP2), bound at the I-site of tDGC, forcing the GGDEF domain in an inhibited conformation, with both A-sites facing away from each other. An omit map (blue mesh) with Fourier coefficients 2Fo-Fc, where the c-di-GMP ligand was omitted from phase calculation is shown at 1σ contour level. Residues from the RxxD motif at the I-site forming hydrogen bonds with the bound c-di-GMP molecules are displayed as sticks and the distances between interacting atoms are displayed. The same color code as in panel a is used.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215984&req=5

pone-0110912-g002: The inactive tDGC dimer (a) The two monomers present in the asymmetric unit are colored in grey and yellow respectively with the two bridging c-di-GMP molecules shown as sticks.The views are along (left) and perpendicular (right) to the non-crystallographic dyad. (b) Stereoview of the dimerization mediated by two c-di-GMP molecules (labeled c-di-GMP1 and c-di-GMP2), bound at the I-site of tDGC, forcing the GGDEF domain in an inhibited conformation, with both A-sites facing away from each other. An omit map (blue mesh) with Fourier coefficients 2Fo-Fc, where the c-di-GMP ligand was omitted from phase calculation is shown at 1σ contour level. Residues from the RxxD motif at the I-site forming hydrogen bonds with the bound c-di-GMP molecules are displayed as sticks and the distances between interacting atoms are displayed. The same color code as in panel a is used.
Mentions: The wild type tDGC protein co-purifies with c-di-GMP and co-crystallizes without any extraneous addition of ligand. The structure was determined at a resolution of 2.27 Å (Tables 1and2). In this crystal form, tDGC adopts a dimeric conformation, where the two monomers are related by a non-crystallographic dyad (Fig. 2). The buried accessible surface area between the two monomers is 1,235 Å2 and their interface is stabilized by one salt bridge and thirteen hydrogen bonds. I-site residues 158-RxxD-161 from the two monomers interact with two molecules of c-di-GMP that are mutually intercalated (Fig. 2a). This symmetrical arrangement is similar to the PleD structure [7], with the four guanyl bases of the two c-di-GMP molecules bound to the primary inhibitory sites of one molecule comprising the 158-RxxD-161 motif and a secondary inhibitory site R115 contributed by the second monomer. Residue R158 plays a key role in the interaction with both c-di-GMP molecules, via its guanidinium moiety that completes the set of stacking interactions (Fig. 2b). In this arrangement, the two monomers are locked in an inactive orientation with their A sites facing away from each other in a catalytically unproductive mode (Fig. 2a). The crystallographic dimer observed in this crystal form is consistent with gel filtration analysis showing that tDGC forms a dimer in solution (Fig. 3a). To demonstrate that dimerization is mediated by c-di-GMP, which co-purifies with tDGC, we incubated the protein with RocR, a PDE from Pseudomonas aeruginosa, to remove the bound c-di-GMP. Remarkably, following enzymatic treatment with RocR, the wild type tDGC protein elutes as a monomer (Fig. 3b).

Bottom Line: Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF domain) from Thermotoga maritima, enables the robust enzymatic production of large quantities of c-di-GMP.To understand the structural correlates of tDGC thermostability, its catalytic mechanism and feedback inhibition, we determined structures of an active-like dimeric conformation with both active (A) sites facing each other and of an inactive dimeric conformation, locked by c-di-GMP bound at the inhibitory (I) site.We also report the structure of a single mutant of tDGC, with the R158A mutation at the I-site, abolishing product inhibition and unproductive dimerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.

ABSTRACT
Large-scale production of bis-3'-5'-cyclic-di-GMP (c-di-GMP) would facilitate biological studies of numerous bacterial signaling pathways and phenotypes controlled by this second messenger molecule, such as virulence and biofilm formation. C-di-GMP constitutes also a potentially interesting molecule as a vaccine adjuvant. Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF domain) from Thermotoga maritima, enables the robust enzymatic production of large quantities of c-di-GMP. To understand the structural correlates of tDGC thermostability, its catalytic mechanism and feedback inhibition, we determined structures of an active-like dimeric conformation with both active (A) sites facing each other and of an inactive dimeric conformation, locked by c-di-GMP bound at the inhibitory (I) site. We also report the structure of a single mutant of tDGC, with the R158A mutation at the I-site, abolishing product inhibition and unproductive dimerization. A comparison with structurally characterized DGC homologues from mesophiles reveals the presence of a higher number of salt bridges in the hyperthermophile enzyme tDGC. Denaturation experiments of mutants disrupting in turn each of the salt bridges unique to tDGC identified three salt-bridges critical to confer thermostability.

Show MeSH