Limits...
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.

Show MeSH
Size Exclusion Chromatograms (a) Gel filtration chromatogram using a S75 column, of the wild type tDGC protein (red) that elutes as a c-di-GMP-bound dimer and of the R158A mutant (green) that purifies as a monomer and is devoid of nucleotides.(b) Following incubation of the wild type tDGC bound to c-di-GMP with RocR (in the gel filtration buffer containing 25 mM Tris-HCl, 300 mM NaCl, 5% (v/v) glycerol, 10 mM MgCl2 for 2 hrs at room temperature), c-diGMP is converted to pGpG and tDGC elutes predominantly as a monomer. SDS PAGE analysis of the dimeric and monomeric fractions demonstrates that both peaks contain tDGC.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215984&req=5

pone-0110912-g003: Size Exclusion Chromatograms (a) Gel filtration chromatogram using a S75 column, of the wild type tDGC protein (red) that elutes as a c-di-GMP-bound dimer and of the R158A mutant (green) that purifies as a monomer and is devoid of nucleotides.(b) Following incubation of the wild type tDGC bound to c-di-GMP with RocR (in the gel filtration buffer containing 25 mM Tris-HCl, 300 mM NaCl, 5% (v/v) glycerol, 10 mM MgCl2 for 2 hrs at room temperature), c-diGMP is converted to pGpG and tDGC elutes predominantly as a monomer. SDS PAGE analysis of the dimeric and monomeric fractions demonstrates that both peaks contain tDGC.

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)

Size Exclusion Chromatograms (a) Gel filtration chromatogram using a S75 column, of the wild type tDGC protein (red) that elutes as a c-di-GMP-bound dimer and of the R158A mutant (green) that purifies as a monomer and is devoid of nucleotides.(b) Following incubation of the wild type tDGC bound to c-di-GMP with RocR (in the gel filtration buffer containing 25 mM Tris-HCl, 300 mM NaCl, 5% (v/v) glycerol, 10 mM MgCl2 for 2 hrs at room temperature), c-diGMP is converted to pGpG and tDGC elutes predominantly as a monomer. SDS PAGE analysis of the dimeric and monomeric fractions demonstrates that both peaks contain tDGC.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110912-g003: Size Exclusion Chromatograms (a) Gel filtration chromatogram using a S75 column, of the wild type tDGC protein (red) that elutes as a c-di-GMP-bound dimer and of the R158A mutant (green) that purifies as a monomer and is devoid of nucleotides.(b) Following incubation of the wild type tDGC bound to c-di-GMP with RocR (in the gel filtration buffer containing 25 mM Tris-HCl, 300 mM NaCl, 5% (v/v) glycerol, 10 mM MgCl2 for 2 hrs at room temperature), c-diGMP is converted to pGpG and tDGC elutes predominantly as a monomer. SDS PAGE analysis of the dimeric and monomeric fractions demonstrates that both peaks contain tDGC.
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