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== Biological Function == | == Biological Function == | ||
[[Image:C-di-GMP larger font.jpg |200 px|thumb|left|'''Figure 1: Cyclic-dimeric-GMP''']] | [[Image:C-di-GMP larger font.jpg |200 px|thumb|left|'''Figure 1: Cyclic-dimeric-GMP.''']] | ||
[[Image:Poly B-1, 6 GlcNAc.jpg |150 px|left|thumb|'''Figure 2: Poly-β-1,6-N-acetylglucosamine''']] | [[Image:Poly B-1, 6 GlcNAc.jpg |150 px|left|thumb|'''Figure 2: Poly-β-1,6-N-acetylglucosamine.''']] | ||
Diguanylate cyclases are a group of class 2 transferase enzymes that catalyze the production of cyclic dimeric-guanosine monophosphate (c-di-GMP), an important <span class="plainlinks">[https://en.wikipedia.org/wiki/Second_messenger_system second messenger]</span> for <span class="plainlinks">[https://en.wikipedia.org/wiki/Signal_transduction signal transduction]</span>.<sup>[1]</sup> Most commonly through phosphorylation or dephosphoylation events, signal transduction sends messages through cells to promote responses. <span class="plainlinks">[https://en.wikipedia.org/wiki/Escherichia_coli ''Escherechia coli'']</span>, a gram-negative bacterium often found in the intestines of mammals, uses diguanylate cyclase DgcZ in the synthesis of its <span class="plainlinks">[https://en.wikipedia.org/wiki/biofilm biofilm]</span>.<sup>[2]</sup> Enzyme DgcZ from ''E. coli'' acts as a catalyst to synthesize cyclic di-GMP from two substrate guanosine triphosphate (GTP) molecules to aid in communication of signals throughout the bacteria. C-di-GMP is a second messenger in the production of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a polysaccharide required for ''E. coli'' biofilm production <sup>[2]</sup>. This biofilm allows ''E. coli'' to adhere to extracellular surfaces. The complete enzyme is only successfully crystallized in its inactive conformation <sup>[3]</sup>. | Diguanylate cyclases are a group of class 2 transferase enzymes that catalyze the production of cyclic dimeric-guanosine monophosphate (c-di-GMP), an important <span class="plainlinks">[https://en.wikipedia.org/wiki/Second_messenger_system second messenger]</span> for <span class="plainlinks">[https://en.wikipedia.org/wiki/Signal_transduction signal transduction]</span>.<sup>[1]</sup> Most commonly through phosphorylation or dephosphoylation events, signal transduction sends messages through cells to promote responses. <span class="plainlinks">[https://en.wikipedia.org/wiki/Escherichia_coli ''Escherechia coli'']</span>, a gram-negative bacterium often found in the intestines of mammals, uses diguanylate cyclase DgcZ in the synthesis of its <span class="plainlinks">[https://en.wikipedia.org/wiki/biofilm biofilm]</span>.<sup>[2]</sup> Enzyme DgcZ from ''E. coli'' acts as a catalyst to synthesize cyclic di-GMP from two substrate guanosine triphosphate (GTP) molecules to aid in communication of signals throughout the bacteria. C-di-GMP is a second messenger in the production of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a polysaccharide required for ''E. coli'' biofilm production <sup>[2]</sup>. This biofilm allows ''E. coli'' to adhere to extracellular surfaces. The complete enzyme is only successfully crystallized in its inactive conformation <sup>[3]</sup>. | ||
[[Image:DgcZ_Conformation_change_1.png|250 px|right|thumb|'''Figure 3: Diagram of DgcZ''' DgcZ is shown in its active (left) and inactive (right) conformations. The boxes represent the GGEEF domains of the enzyme, while the cylinders represent the alpha helices of the CZB domain, which contains the Zinc binding sites. Binding Zn<sup>+2</sup> inactivates the enzyme. The red vs blue coloring represents the symmetry of the enzyme.]] | [[Image:DgcZ_Conformation_change_1.png|250 px|right|thumb|'''Figure 3: Diagram of DgcZ''' DgcZ is shown in its active (left) and inactive (right) conformations. The boxes represent the GGEEF domains of the enzyme, while the cylinders represent the alpha helices of the CZB domain, which contains the Zinc binding sites. Binding Zn<sup>+2</sup> inactivates the enzyme. The red vs blue coloring represents the symmetry of the enzyme.]] | ||
== Structural Overview == | == Structural Overview == | ||
[[Image:DgcZ full molecule all sites and ligands labeled.png|250 px|left|thumb|'''Figure 4: Diguanylate cyclase DgcZ from “E. Coli”''' The two domains of the enzyme are labeled]] | [[Image:DgcZ full molecule all sites and ligands labeled.png|250 px|left|thumb|'''Figure 4: Diguanylate cyclase DgcZ from “E. Coli”.''' The two domains of the enzyme are labeled]] | ||
DgcZ is a dimeric protein from ''E. coli'' made of <scene name='69/694239/Dgcz_ggeef_dom_and_czb_dom/1'>two domains</scene> <sup>[4]</sup>. The DgcZ protein has <scene name='69/694239/C2_symmetry/6'>C2</scene> symmetry down its central axis. DgcZ has <scene name='69/694239/Dgcz_ggeef_dom_and_czb_dom/1'>two domains</scene> <sup>[4]</sup>. The catalytic glycine-glycine-glutamate-glutamate-phenylalanine (GGEEF) domain is responsible for synthesizing c-di-GMP, and the regulatory chemoreceptor zinc binding (CZB) domain houses two zinc binding sites. DgcZ binds zinc in the CZB domain with sub-femtomolar (10<sup>-16</sup>M) 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. CZB and GGEEF domains are prevalent in many bacterial proteins from differing strands of ''E. coli'' <sup>[2]</sup>. 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. | DgcZ is a dimeric protein from ''E. coli'' made of <scene name='69/694239/Dgcz_ggeef_dom_and_czb_dom/1'>two domains</scene> <sup>[4]</sup>. The DgcZ protein has <scene name='69/694239/C2_symmetry/6'>C2</scene> symmetry down its central axis. DgcZ has <scene name='69/694239/Dgcz_ggeef_dom_and_czb_dom/1'>two domains</scene> <sup>[4]</sup>. The catalytic glycine-glycine-glutamate-glutamate-phenylalanine (GGEEF) domain is responsible for synthesizing c-di-GMP, and the regulatory chemoreceptor zinc binding (CZB) domain houses two zinc binding sites. DgcZ binds zinc in the CZB domain with sub-femtomolar (10<sup>-16</sup>M) 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. CZB and GGEEF domains are prevalent in many bacterial proteins from differing strands of ''E. coli'' <sup>[2]</sup>. 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. | ||
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==CZB Domain and Zinc Binding Site== | ==CZB Domain and Zinc Binding Site== | ||
The <scene name='69/694239/Zb_domain_residues_19-90/6'>CZB domain</scene>, residues 19-90, is responsible for regulating the function of DgcZ due to the presence of two zinc binding sites. The domain contains the <scene name='69/694239/Zinc_binding_domain_zmout/1'>allosteric binding sites</scene> of the enzyme which exhibits 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 complete enzyme has not yet been crystallized in its 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. | The <scene name='69/694239/Zb_domain_residues_19-90/6'>CZB domain</scene>, residues 19-90, is responsible for regulating the function of DgcZ due to the presence of two zinc binding sites. The domain contains the <scene name='69/694239/Zinc_binding_domain_zmout/1'>allosteric binding sites</scene> of the enzyme which exhibits 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 complete enzyme has not yet been crystallized in its 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|'''Figure 5: Zn<sup>+2</sup> Coordination to amino acid residues''' Three of the four 𝝰 helices of the CZB domain of DgcZ coordinate to the Zn<sup>+2</sup> ion for binding.]] | [[Image:Zinc binding site labels.jpg|250 px|left|thumb|'''Figure 5: Zn<sup>+2</sup> Coordination to amino acid residues.''' Three of the four 𝝰 helices of the CZB domain of DgcZ coordinate to the Zn<sup>+2</sup> ion for binding.]] | ||
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_zm_in/1'>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 <scene name='69/694239/Ntermctermdgcz/1'>clarification</scene>, 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. | 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_zm_in/1'>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 <scene name='69/694239/Ntermctermdgcz/1'>clarification</scene>, 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. | ||