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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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Temperature-dependent unfolding of tDGC and of tDGC single mutants recorded by CD spectropolarimetry.The mean residue molar ellipticity vs temperature for proteins tDGC (dimeric form), tDGC (monomeric form, following treatment with RocR), D177A, E196A and R233A is plotted. A significant difference in the melting profiles of the wild type and the single tDGC mutants (disrupting individual salt bridge mutants) is visible. The melting temperature Tm, was determined by a Boltzmann sigmoid analysis (see text).
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pone-0110912-g006: Temperature-dependent unfolding of tDGC and of tDGC single mutants recorded by CD spectropolarimetry.The mean residue molar ellipticity vs temperature for proteins tDGC (dimeric form), tDGC (monomeric form, following treatment with RocR), D177A, E196A and R233A is plotted. A significant difference in the melting profiles of the wild type and the single tDGC mutants (disrupting individual salt bridge mutants) is visible. The melting temperature Tm, was determined by a Boltzmann sigmoid analysis (see text).

Mentions: To analyze the contribution of each salt bridge to tDGC thermostability, single mutants D177A, E196A and R233A were made to disrupt the three unique solvent exposed salt-bridges (Fig. 5b). The three mutants eluted as dimers in gel filtration, indicating that c-di-GMP is bound to their I-site. To evaluate the stabilization effects brought about by nucleotide binding and dimerization in the thermal denaturation experiments, the nucleotide-free monomeric form of the wild type protein was generated by incubating with RocR [23], in order to digest the c-di-GMP molecule bridging the two tDGC monomers. This digestion step was followed by gel filtration to remove RocR and pGpG. The melting temperatures of the wild type tDGC and the mutants were measured by circular dichroism at 220 nm (Fig. 6). The monomeric and dimeric forms of the wild type tDGC protein exhibit comparable melting temperatures with Tm values of 85.5°C and 85.0°C respectively. Disruption of the R152-E196 salt bridge has the smallest effect on tDGC thermostability (decrease of 4.2°C). However, disruption of the other two salt bridges, K118-D177 and D219-R233, lead to large reductions in the melting temperature of tDGC by 12.3°C and 8.6°C, respectively (Fig. 6).


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)

Temperature-dependent unfolding of tDGC and of tDGC single mutants recorded by CD spectropolarimetry.The mean residue molar ellipticity vs temperature for proteins tDGC (dimeric form), tDGC (monomeric form, following treatment with RocR), D177A, E196A and R233A is plotted. A significant difference in the melting profiles of the wild type and the single tDGC mutants (disrupting individual salt bridge mutants) is visible. The melting temperature Tm, was determined by a Boltzmann sigmoid analysis (see text).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110912-g006: Temperature-dependent unfolding of tDGC and of tDGC single mutants recorded by CD spectropolarimetry.The mean residue molar ellipticity vs temperature for proteins tDGC (dimeric form), tDGC (monomeric form, following treatment with RocR), D177A, E196A and R233A is plotted. A significant difference in the melting profiles of the wild type and the single tDGC mutants (disrupting individual salt bridge mutants) is visible. The melting temperature Tm, was determined by a Boltzmann sigmoid analysis (see text).
Mentions: To analyze the contribution of each salt bridge to tDGC thermostability, single mutants D177A, E196A and R233A were made to disrupt the three unique solvent exposed salt-bridges (Fig. 5b). The three mutants eluted as dimers in gel filtration, indicating that c-di-GMP is bound to their I-site. To evaluate the stabilization effects brought about by nucleotide binding and dimerization in the thermal denaturation experiments, the nucleotide-free monomeric form of the wild type protein was generated by incubating with RocR [23], in order to digest the c-di-GMP molecule bridging the two tDGC monomers. This digestion step was followed by gel filtration to remove RocR and pGpG. The melting temperatures of the wild type tDGC and the mutants were measured by circular dichroism at 220 nm (Fig. 6). The monomeric and dimeric forms of the wild type tDGC protein exhibit comparable melting temperatures with Tm values of 85.5°C and 85.0°C respectively. Disruption of the R152-E196 salt bridge has the smallest effect on tDGC thermostability (decrease of 4.2°C). However, disruption of the other two salt bridges, K118-D177 and D219-R233, lead to large reductions in the melting temperature of tDGC by 12.3°C and 8.6°C, respectively (Fig. 6).

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