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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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Domain architecture of tDGC.(a) Topology of the GGDEF domain of tDGC. The N- and C- termini of the polypeptide chain are indicated. The β-strands are depicted by pink arrows and α-helices by blue tubes. (b) Location of the A- and I-site on the structure of tDGC: The loops bearing the “GGDEF” motif at the A-site and “RxxD” motif at the I-site are shown in red and blue respectively. (c) Superposition of the A site of tDGC (cyan) and PleD (dark blue)7. GTPαS-Mg2+, bound to PleD is shown as sticks and green spheres respectively. Residues involved in metal ion binding and base recognition (represented as sticks and labeled according to tDGC and PleD numbering schemes) are strictly conserved between the two proteins.
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pone-0110912-g001: Domain architecture of tDGC.(a) Topology of the GGDEF domain of tDGC. The N- and C- termini of the polypeptide chain are indicated. The β-strands are depicted by pink arrows and α-helices by blue tubes. (b) Location of the A- and I-site on the structure of tDGC: The loops bearing the “GGDEF” motif at the A-site and “RxxD” motif at the I-site are shown in red and blue respectively. (c) Superposition of the A site of tDGC (cyan) and PleD (dark blue)7. GTPαS-Mg2+, bound to PleD is shown as sticks and green spheres respectively. Residues involved in metal ion binding and base recognition (represented as sticks and labeled according to tDGC and PleD numbering schemes) are strictly conserved between the two proteins.

Mentions: We expressed and crystallized a domain of the TM1788 protein from Thermotoga maritima (248 residues, WP_010865403) spanning residues 82–248, hereafter named tDGC. The structures of tDGC and of its R158A mutant were determined in three crystal forms (i) tDGC in an active-like dimeric conformation (ii) tDGC in an inactive dimeric conformation (iii) R158A mutant in a monomeric conformation (Tables 1 and 2). The overall structure of the tDGC monomer features a central antiparallel β-sheet with the topology β2-β3-β1-β6, surrounded by five α-helices (Fig. 1a). The location of the conserved catalytic 167-GGDEF-171 motif (A-site) and the inhibitory I-site (158-RxxD-161) on loops connecting β2 and β3 and between α3 and β2 respectively, are depicted in Fig. 1b. tDGC shares a fold similar to adenylyl cyclases and the palm domain of polymerases, with a reaction mechanism involving two metal ions, which are observed in the structure of PleD, crystallized with GTPαS, but not in the present structures. According to this reaction scheme, Asp169 deprotonates the 3′ hydroxyl group of the ribose of the GTP substrate to initiate an in-line nucleophilic attack on the α-phosphate of the target GTP, followed by elimination of the pyrophosphate moiety (Fig. 1c). The four residues responsible for coordinating the Mg2+ ion (Asp126 and Asp169) and for making contact with the guanine moiety (Asn134 and Asp143) are strictly conserved in tDGC, as shown in Fig. 1c.


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)

Domain architecture of tDGC.(a) Topology of the GGDEF domain of tDGC. The N- and C- termini of the polypeptide chain are indicated. The β-strands are depicted by pink arrows and α-helices by blue tubes. (b) Location of the A- and I-site on the structure of tDGC: The loops bearing the “GGDEF” motif at the A-site and “RxxD” motif at the I-site are shown in red and blue respectively. (c) Superposition of the A site of tDGC (cyan) and PleD (dark blue)7. GTPαS-Mg2+, bound to PleD is shown as sticks and green spheres respectively. Residues involved in metal ion binding and base recognition (represented as sticks and labeled according to tDGC and PleD numbering schemes) are strictly conserved between the two proteins.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110912-g001: Domain architecture of tDGC.(a) Topology of the GGDEF domain of tDGC. The N- and C- termini of the polypeptide chain are indicated. The β-strands are depicted by pink arrows and α-helices by blue tubes. (b) Location of the A- and I-site on the structure of tDGC: The loops bearing the “GGDEF” motif at the A-site and “RxxD” motif at the I-site are shown in red and blue respectively. (c) Superposition of the A site of tDGC (cyan) and PleD (dark blue)7. GTPαS-Mg2+, bound to PleD is shown as sticks and green spheres respectively. Residues involved in metal ion binding and base recognition (represented as sticks and labeled according to tDGC and PleD numbering schemes) are strictly conserved between the two proteins.
Mentions: We expressed and crystallized a domain of the TM1788 protein from Thermotoga maritima (248 residues, WP_010865403) spanning residues 82–248, hereafter named tDGC. The structures of tDGC and of its R158A mutant were determined in three crystal forms (i) tDGC in an active-like dimeric conformation (ii) tDGC in an inactive dimeric conformation (iii) R158A mutant in a monomeric conformation (Tables 1 and 2). The overall structure of the tDGC monomer features a central antiparallel β-sheet with the topology β2-β3-β1-β6, surrounded by five α-helices (Fig. 1a). The location of the conserved catalytic 167-GGDEF-171 motif (A-site) and the inhibitory I-site (158-RxxD-161) on loops connecting β2 and β3 and between α3 and β2 respectively, are depicted in Fig. 1b. tDGC shares a fold similar to adenylyl cyclases and the palm domain of polymerases, with a reaction mechanism involving two metal ions, which are observed in the structure of PleD, crystallized with GTPαS, but not in the present structures. According to this reaction scheme, Asp169 deprotonates the 3′ hydroxyl group of the ribose of the GTP substrate to initiate an in-line nucleophilic attack on the α-phosphate of the target GTP, followed by elimination of the pyrophosphate moiety (Fig. 1c). The four residues responsible for coordinating the Mg2+ ion (Asp126 and Asp169) and for making contact with the guanine moiety (Asn134 and Asp143) are strictly conserved in tDGC, as shown in Fig. 1c.

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