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== Structural Overview == | == Structural Overview == | ||
[[Image:DgcZ full molecule all sites and ligands labeled.png|250 px|left|thumb|Diguanylate cyclase DgcZ from “E. Coli” with Domains Labeled]] | [[Image:DgcZ full molecule all sites and ligands labeled.png|250 px|left|thumb|Diguanylate cyclase DgcZ from “E. Coli” with Domains Labeled]] | ||
The DgcZ protein has C2 symmetry composed of two domains. The catalytic glycine-glycine-glutamate-glutamate-phenylalanine (GGEEF) domain responsible for synthesizing c-di-GMP and the regulatory chemoreceptor zinc binding (CZB) domain comprising two zinc binding sites. DgcZ binds zinc with sub-femtomolar affinity. When zinc is bound, the CZB and GGEEF domains adopt conformations that inhibit DgcZ function <sup>[1]</sup>. Enzyme DgcZ was co-crystallized with Zinc fixing the structure in its inactivate conformation. The CZB domain is common to many bacterial lineages, including its prevalence in DgcZ homologs. The domain has an important role in signal transduction of bacteria. Many bacterial proteins from differing strands of ''E. coli'' contain CZB and GGEEF domains<sup>[2]</sup>. ''E. coli'' DgcZ is a protein made of two domains each of which is a symmetric homodimer. It exhibits <scene name='69/694239/C2_symmetry/6'>C2</scene> symmetry down its central axis. The GGEEF domain is catalytic in that it contains the active sites used for cyclizing GTP into c-di-GMP. The CZB domain is used for ligand-mediated regulation of c-di-GMP production | The DgcZ protein has C2 symmetry composed of two domains. The catalytic glycine-glycine-glutamate-glutamate-phenylalanine (GGEEF) domain responsible for synthesizing c-di-GMP and the regulatory chemoreceptor zinc binding (CZB) domain comprising two zinc binding sites. DgcZ binds zinc with sub-femtomolar affinity. When zinc is bound, the CZB and GGEEF domains adopt conformations that inhibit DgcZ function <sup>[1]</sup>. Enzyme DgcZ was co-crystallized with Zinc fixing the structure in its inactivate conformation. The CZB domain is common to many bacterial lineages, including its prevalence in DgcZ homologs. The domain has an important role in signal transduction of bacteria. Many bacterial proteins from differing strands of ''E. coli'' contain CZB and GGEEF domains<sup>[2]</sup>. ''E. coli'' DgcZ is a protein made of two domains each of which is a symmetric homodimer. It exhibits <scene name='69/694239/C2_symmetry/6'>C2</scene> symmetry down its central axis. The GGEEF domain is catalytic in that it contains the active sites used for cyclizing GTP into c-di-GMP. The CZB domain is used for ligand-mediated regulation of c-di-GMP production. | ||
===Catalytic GGEEF Domain=== | ===Catalytic GGEEF Domain=== | ||
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[[Image:Electrostatic map front view CZB.png|250 px|right|thumb|Electrostatic potential map of the CZB domain of Diguanylate Cyclase. Regions of relatively negative charge are in red and regions of relatively positive charge are in blue. Electrically neutral regions are in white]] | [[Image:Electrostatic map front view CZB.png|250 px|right|thumb|Electrostatic potential map of the CZB domain of Diguanylate Cyclase. Regions of relatively negative charge are in red and regions of relatively positive charge are in blue. Electrically neutral regions are in white]] | ||
=== Zinc Binding Site === | === Zinc Binding Site === | ||
Most cells possess efficient Zinc uptake systems, as Zinc is a reactive Lewis Acid. Zinc binds incredibly tightly to this enzyme at subfemtomolar concentrations. The Zinc co-purified with the protein.Zinc allosterically inhibits the activity of enzyme DgcZ through two allosteric binding sites located on the CZB domain. The inhibition prevents regulation of GGDEF domain function, the location of the active site. The CZB domain is folded into four anti-parallel α-helices as a 2-fold symmetric homodimer, with the N-terminus on the helix 𝝰4. The allosteric binding site includes a <scene name='69/694239/Zinc_binding_domain/4'>3His/1Cys</scene> motif that uses amino acids H22 of 𝝰1, C52 of 𝝰2, and H79 and H83 of 𝝰3, spanning three of the four alpha helices of the CZB domain and coordinating the Zinc residue in a tetrahedral fashion. For clarification, the entirety of 𝝰helix 2 on one monomer of CZB is not successfully crystallized after the Cys52 reside and is not the N-terminal residue. | Most cells possess efficient Zinc uptake systems, as Zinc is a reactive Lewis Acid. Zinc binds incredibly tightly to this enzyme at subfemtomolar concentrations. The Zinc co-purified with the protein.Zinc allosterically inhibits the activity of enzyme DgcZ through two allosteric binding sites located on the CZB domain <sup>[1]</sup>. The inhibition prevents regulation of GGDEF domain function, the location of the active site. The CZB domain is folded into four anti-parallel α-helices as a 2-fold symmetric homodimer, with the N-terminus on the helix 𝝰4. The allosteric binding site includes a <scene name='69/694239/Zinc_binding_domain/4'>3His/1Cys</scene> motif that uses amino acids H22 of 𝝰1, C52 of 𝝰2, and H79 and H83 of 𝝰3, spanning three of the four alpha helices of the CZB domain and coordinating the Zinc residue in a tetrahedral fashion. For clarification, the entirety of 𝝰helix 2 on one monomer of CZB is not successfully crystallized after the Cys52 reside and is not the N-terminal residue. | ||
Zahringer et al. mutated Cys52 to Ala through <span class="plainlinks">[https://en.wikipedia.org/wiki/Site-directed_mutagenesis site-directed mutagenesis]</span>, resulting in a lack of coordination on α2. The cysteine residue is not essential for Zinc binding, as Zinc still coordinates to the three His residues with the Cys52Ala mutation, but α2 is free to move and expose the Zinc binding pocket. This exposure was found to lower the protein's affinity for zinc, as the mutation of cysteine to alanine increased the activity of the DgcZ. Using EDTA, Zinc can be removed from the CZB domain. The zinc has higher affinity for EDTA than CZB when EDTA concentration is higher than the concentration of DgcZ. When not coordinated to zinc, the CZB domain adopts a conformation that straightens the <scene name='69/694239/Czbd_with_helices_labeled/2'>𝝰1 helix</scene>, shifting <scene name='69/694239/Hydrophobicity_int_residues/3'>hydrophobic residues</scene> on the α-helices into the center and the GGEEF domain into its productive conformation, increasing activity of DgcZ. Activity increases without Zinc due to activation of poly-GlcNAc production and biofilm formation, and maximal cyclic di-GMP production. | Zahringer et al. mutated Cys52 to Ala through <span class="plainlinks">[https://en.wikipedia.org/wiki/Site-directed_mutagenesis site-directed mutagenesis]</span>, resulting in a lack of coordination on α2. The cysteine residue is not essential for Zinc binding, as Zinc still coordinates to the three His residues with the Cys52Ala mutation, but α2 is free to move and expose the Zinc binding pocket. This exposure was found to lower the protein's affinity for zinc, as the mutation of cysteine to alanine increased the activity of the DgcZ. Using EDTA, Zinc can be removed from the CZB domain. The zinc has higher affinity for EDTA than CZB when EDTA concentration is higher than the concentration of DgcZ. When not coordinated to zinc, the CZB domain adopts a conformation that straightens the <scene name='69/694239/Czbd_with_helices_labeled/2'>𝝰1 helix</scene>, shifting <scene name='69/694239/Hydrophobicity_int_residues/3'>hydrophobic residues</scene> on the α-helices into the center and the GGEEF domain into its productive conformation, increasing activity of DgcZ. Activity increases without Zinc due to activation of poly-GlcNAc production and biofilm formation, and maximal cyclic di-GMP production. | ||