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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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Structure-based sequence alignment of selected DGCs and salt-bridges unique to tDGC.(a) Sequence alignment between tDGC from Thermotoga maritima (this work), mesophilic DGC domains (3ICL, 4IOB, 3IGN and 3EZU). The sequence of the structurally characterized PleD (2V0N) from Caulobacter crescentus is also included for comparison. The secondary structure elements of tDGC are displayed above the sequences. Strictly conserved residues are in red boxes and chemically similar residues are colored in red. Residues D126 and D169 of tDGC are involved in divalent metal coordination. The A- and I-site residues are highlighted with green and black triangles respectively. The blue triangles indicate salt-bridge forming residues that are conserved across the five proteins. Red triangles indicate residues unique to tDGC involved in salt bridge formation. (b) Mapping of the salt bridges unique to the thermostable enzyme onto the 3D structure of tDGC: Lys118-Asp177, Arg233-Asp219 and Arg152–Glu196 were disrupted in the site-directed mutagenesis study.
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pone-0110912-g005: Structure-based sequence alignment of selected DGCs and salt-bridges unique to tDGC.(a) Sequence alignment between tDGC from Thermotoga maritima (this work), mesophilic DGC domains (3ICL, 4IOB, 3IGN and 3EZU). The sequence of the structurally characterized PleD (2V0N) from Caulobacter crescentus is also included for comparison. The secondary structure elements of tDGC are displayed above the sequences. Strictly conserved residues are in red boxes and chemically similar residues are colored in red. Residues D126 and D169 of tDGC are involved in divalent metal coordination. The A- and I-site residues are highlighted with green and black triangles respectively. The blue triangles indicate salt-bridge forming residues that are conserved across the five proteins. Red triangles indicate residues unique to tDGC involved in salt bridge formation. (b) Mapping of the salt bridges unique to the thermostable enzyme onto the 3D structure of tDGC: Lys118-Asp177, Arg233-Asp219 and Arg152–Glu196 were disrupted in the site-directed mutagenesis study.

Mentions: The thermostable GGDEF domain of tDGC shares a conserved structure with homologous proteins from three mesophilic species: Pseudomonas aeruginosa, Marinobacter aquaeolei and Geobacter sulfurreducens (Table 1 in File S1) and one thermo-tolerant organism Methylococcus capsulatus16. Several molecular factors that potentially contribute to thermostability were examined (Table 1 in File S1). This comparison revealed that the increased thermostability of tDGC could be predominantly due to an excess of salt bridges (total of 5) compared to mesophilic homologues (1 or 2) (Table 2 in File S1). Only salt bridges that connect residues far apart in the tDGC amino-acid sequence were considered, as protein unfolding would not disrupt salt bridges involving residues adjacent in the sequence. A sequence alignment (Fig. 5a) reveals that of the five salt bridges present, two are conserved across protein homologues (D91-R165 and D126-K238), whilst the three salt bridges: K118-D177, R152-E196 and R233-D219 (Fig. 5) are unique to the thermostable enzyme. This is consistent with the observation that several Thermotoga maritima proteins possess an excess of salt bridges, compared to their mesophilic counterparts [31], [32].


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)

Structure-based sequence alignment of selected DGCs and salt-bridges unique to tDGC.(a) Sequence alignment between tDGC from Thermotoga maritima (this work), mesophilic DGC domains (3ICL, 4IOB, 3IGN and 3EZU). The sequence of the structurally characterized PleD (2V0N) from Caulobacter crescentus is also included for comparison. The secondary structure elements of tDGC are displayed above the sequences. Strictly conserved residues are in red boxes and chemically similar residues are colored in red. Residues D126 and D169 of tDGC are involved in divalent metal coordination. The A- and I-site residues are highlighted with green and black triangles respectively. The blue triangles indicate salt-bridge forming residues that are conserved across the five proteins. Red triangles indicate residues unique to tDGC involved in salt bridge formation. (b) Mapping of the salt bridges unique to the thermostable enzyme onto the 3D structure of tDGC: Lys118-Asp177, Arg233-Asp219 and Arg152–Glu196 were disrupted in the site-directed mutagenesis study.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110912-g005: Structure-based sequence alignment of selected DGCs and salt-bridges unique to tDGC.(a) Sequence alignment between tDGC from Thermotoga maritima (this work), mesophilic DGC domains (3ICL, 4IOB, 3IGN and 3EZU). The sequence of the structurally characterized PleD (2V0N) from Caulobacter crescentus is also included for comparison. The secondary structure elements of tDGC are displayed above the sequences. Strictly conserved residues are in red boxes and chemically similar residues are colored in red. Residues D126 and D169 of tDGC are involved in divalent metal coordination. The A- and I-site residues are highlighted with green and black triangles respectively. The blue triangles indicate salt-bridge forming residues that are conserved across the five proteins. Red triangles indicate residues unique to tDGC involved in salt bridge formation. (b) Mapping of the salt bridges unique to the thermostable enzyme onto the 3D structure of tDGC: Lys118-Asp177, Arg233-Asp219 and Arg152–Glu196 were disrupted in the site-directed mutagenesis study.
Mentions: The thermostable GGDEF domain of tDGC shares a conserved structure with homologous proteins from three mesophilic species: Pseudomonas aeruginosa, Marinobacter aquaeolei and Geobacter sulfurreducens (Table 1 in File S1) and one thermo-tolerant organism Methylococcus capsulatus16. Several molecular factors that potentially contribute to thermostability were examined (Table 1 in File S1). This comparison revealed that the increased thermostability of tDGC could be predominantly due to an excess of salt bridges (total of 5) compared to mesophilic homologues (1 or 2) (Table 2 in File S1). Only salt bridges that connect residues far apart in the tDGC amino-acid sequence were considered, as protein unfolding would not disrupt salt bridges involving residues adjacent in the sequence. A sequence alignment (Fig. 5a) reveals that of the five salt bridges present, two are conserved across protein homologues (D91-R165 and D126-K238), whilst the three salt bridges: K118-D177, R152-E196 and R233-D219 (Fig. 5) are unique to the thermostable enzyme. This is consistent with the observation that several Thermotoga maritima proteins possess an excess of salt bridges, compared to their mesophilic counterparts [31], [32].

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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