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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 active-like tDGC dimer and the cyclization reaction.(a) Structure of tDGC crystallized in a dimeric active-like conformation, with the two half active sites (loops containing the GGDEF motif colored in blue) facing each other. One monomer is colored yellow and the other grey. The single c-di-GMP molecule bound to the I-site of the monomer colored in yellow, is shown as sticks. (b) “Optimized” dimer where the residual transformation (Figure S2 in File S1) was applied to the monomer colored grey to generate a dimer with exact two-fold symmetry. The view is along the non-crystallographic dyad that runs across the two GTP molecules displayed as sticks. (c) Magnified view of the tDGC-GTP-Mg Michaelis complex, modeled on the basis of the “optimized” 2-fold symmetric dimer. The arrows indicate the nucleophilic attack of the 3′ oxygen atom on the α-phosphate of the adjacent GTP. (d) Superposition of the “optimized” active dimer (this work, yellow and grey) and the c-diGMP cross-linked YdeH dimer (PDB code: 3TVK, Zaehringer and Schirmer) (colored in pink and teal).
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pone-0110912-g004: The active-like tDGC dimer and the cyclization reaction.(a) Structure of tDGC crystallized in a dimeric active-like conformation, with the two half active sites (loops containing the GGDEF motif colored in blue) facing each other. One monomer is colored yellow and the other grey. The single c-di-GMP molecule bound to the I-site of the monomer colored in yellow, is shown as sticks. (b) “Optimized” dimer where the residual transformation (Figure S2 in File S1) was applied to the monomer colored grey to generate a dimer with exact two-fold symmetry. The view is along the non-crystallographic dyad that runs across the two GTP molecules displayed as sticks. (c) Magnified view of the tDGC-GTP-Mg Michaelis complex, modeled on the basis of the “optimized” 2-fold symmetric dimer. The arrows indicate the nucleophilic attack of the 3′ oxygen atom on the α-phosphate of the adjacent GTP. (d) Superposition of the “optimized” active dimer (this work, yellow and grey) and the c-diGMP cross-linked YdeH dimer (PDB code: 3TVK, Zaehringer and Schirmer) (colored in pink and teal).

Mentions: The structure of tDGC at pH 4, in an alternative dimeric form, was determined to a resolution of 1.9 Å (Tables 1and2). In this crystal form, two c-di-GMP molecules are bound to the I-site of a single tDGC monomer and the A-sites are brought close to each other (Fig. 4a). The absence of a second c-di-GMP molecule bound to the other monomer of the asymmetric unit, eliminates the locking of the monomers in a non-productive orientation. The buried interface area between the two monomers is 905 Å2. In this novel dimeric arrangement, the two monomers are related by a rotation of 167°, a value close to the transformation thought to lead to an active 2-fold symmetric dimer, poised to react with two GTP substrate molecules to perform the cyclization reaction [7] (Fig. 4a). According to the postulated mechanism for the formation of c-di-GMP, each A-site binds to a GTP molecule to form a 2-fold symmetric active dimer with the A sites in proximity [7]. Despite several attempts using soaking or cocrystallization, we could not observe bound GTPαS in this crystal form. The crystals were obtained at pH of 4 at which the carboxylic groups of the aspartate residues involved in metal binding and base recognition (Fig. 1c) are likely to be protonated. The rather acidic conditions of crystallization are therefore likely to hamper metal and substrate binding although the protein conformation looks active (Fig. 4). The structure of PleD7 bound to GTPαS was therefore used as a guide to position two GTP molecules in an optimized 2-fold symmetric dimer (Fig. 4b). Only minor structural movements are then required to positions the α-phosphate groups of the two GTP molecules near the 3′oxygen atoms of the ribose of the adjacent GTP, showing how nucleophilic attack can initiate the cyclization reaction (Fig. 4c).


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 active-like tDGC dimer and the cyclization reaction.(a) Structure of tDGC crystallized in a dimeric active-like conformation, with the two half active sites (loops containing the GGDEF motif colored in blue) facing each other. One monomer is colored yellow and the other grey. The single c-di-GMP molecule bound to the I-site of the monomer colored in yellow, is shown as sticks. (b) “Optimized” dimer where the residual transformation (Figure S2 in File S1) was applied to the monomer colored grey to generate a dimer with exact two-fold symmetry. The view is along the non-crystallographic dyad that runs across the two GTP molecules displayed as sticks. (c) Magnified view of the tDGC-GTP-Mg Michaelis complex, modeled on the basis of the “optimized” 2-fold symmetric dimer. The arrows indicate the nucleophilic attack of the 3′ oxygen atom on the α-phosphate of the adjacent GTP. (d) Superposition of the “optimized” active dimer (this work, yellow and grey) and the c-diGMP cross-linked YdeH dimer (PDB code: 3TVK, Zaehringer and Schirmer) (colored in pink and teal).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215984&req=5

pone-0110912-g004: The active-like tDGC dimer and the cyclization reaction.(a) Structure of tDGC crystallized in a dimeric active-like conformation, with the two half active sites (loops containing the GGDEF motif colored in blue) facing each other. One monomer is colored yellow and the other grey. The single c-di-GMP molecule bound to the I-site of the monomer colored in yellow, is shown as sticks. (b) “Optimized” dimer where the residual transformation (Figure S2 in File S1) was applied to the monomer colored grey to generate a dimer with exact two-fold symmetry. The view is along the non-crystallographic dyad that runs across the two GTP molecules displayed as sticks. (c) Magnified view of the tDGC-GTP-Mg Michaelis complex, modeled on the basis of the “optimized” 2-fold symmetric dimer. The arrows indicate the nucleophilic attack of the 3′ oxygen atom on the α-phosphate of the adjacent GTP. (d) Superposition of the “optimized” active dimer (this work, yellow and grey) and the c-diGMP cross-linked YdeH dimer (PDB code: 3TVK, Zaehringer and Schirmer) (colored in pink and teal).
Mentions: The structure of tDGC at pH 4, in an alternative dimeric form, was determined to a resolution of 1.9 Å (Tables 1and2). In this crystal form, two c-di-GMP molecules are bound to the I-site of a single tDGC monomer and the A-sites are brought close to each other (Fig. 4a). The absence of a second c-di-GMP molecule bound to the other monomer of the asymmetric unit, eliminates the locking of the monomers in a non-productive orientation. The buried interface area between the two monomers is 905 Å2. In this novel dimeric arrangement, the two monomers are related by a rotation of 167°, a value close to the transformation thought to lead to an active 2-fold symmetric dimer, poised to react with two GTP substrate molecules to perform the cyclization reaction [7] (Fig. 4a). According to the postulated mechanism for the formation of c-di-GMP, each A-site binds to a GTP molecule to form a 2-fold symmetric active dimer with the A sites in proximity [7]. Despite several attempts using soaking or cocrystallization, we could not observe bound GTPαS in this crystal form. The crystals were obtained at pH of 4 at which the carboxylic groups of the aspartate residues involved in metal binding and base recognition (Fig. 1c) are likely to be protonated. The rather acidic conditions of crystallization are therefore likely to hamper metal and substrate binding although the protein conformation looks active (Fig. 4). The structure of PleD7 bound to GTPαS was therefore used as a guide to position two GTP molecules in an optimized 2-fold symmetric dimer (Fig. 4b). Only minor structural movements are then required to positions the α-phosphate groups of the two GTP molecules near the 3′oxygen atoms of the ribose of the adjacent GTP, showing how nucleophilic attack can initiate the cyclization reaction (Fig. 4c).

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
Related in: MedlinePlus