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===CZB Domain=== | ===CZB Domain=== | ||
The <scene name='69/694239/Zb_domain_residues_19-90/4'>CZB Domain</scene>, residues 19-90, is responsible for regulating the function of DgcZ. The domain contains the allosteric binding site of the enzyme with cooperative binding. Four residues bind zinc with a high affinity even at 10<sup>-16</sup>M concentrations. Due to the tightness of Zinc binding, the enzyme has not yet been crystallized in | The <scene name='69/694239/Zb_domain_residues_19-90/4'>CZB Domain</scene>, residues 19-90, is responsible for regulating the function of DgcZ. The domain contains the allosteric binding site of the enzyme with cooperative binding. Four residues bind zinc with a high affinity even at 10<sup>-16</sup>M concentrations of Zinc in solution. Due to the tightness of Zinc binding, the enzyme has not yet been crystallized in its complete active conformation without the presence of Zinc metal inhibitor. When zinc is bound, DgcZ activity is limited<sup>[1]</sup>. Two Zinc binding sites are located on the CZB domain. | ||
[[Image:Zinc binding site labels.jpg|250 px|left|thumb|Zn<sup>+2</sup> Coordination to amino acid residues on three of the four 𝝰 helices of DgcZ]] | [[Image:Zinc binding site labels.jpg|250 px|left|thumb|Zn<sup>+2</sup> Coordination to amino acid residues on three of the four 𝝰 helices of DgcZ]] | ||
[[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, attributing to why the enzyme has not been crystallized without Zinc present. 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 | Most cells possess efficient Zinc uptake systems, as Zinc is a reactive Lewis Acid. Zinc binds incredibly tightly to this enzyme at subfemtomolar concentrations, attributing to why the enzyme has not been crystallized without Zinc present. 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 residue 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. | 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. When not coordinated to zinc, the CZB domain presumably 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. | ||
=== Other Ligands === | === Other Ligands === | ||