Sandbox Reserved 1072: Difference between revisions
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[[Image:Poly B-1, 6 GlcNAc.jpg |175 px|left|thumb|'''Figure 2: Poly-β-1,6-N-acetylglucosamine.''' Poly-β-1,6-N-acetylglucosamine is a carbohydrate formed downstream of c-di-GMP utilized in formation of bacterial bio-films.]] | [[Image:Poly B-1, 6 GlcNAc.jpg |175 px|left|thumb|'''Figure 2: Poly-β-1,6-N-acetylglucosamine.''' Poly-β-1,6-N-acetylglucosamine is a carbohydrate formed downstream of c-di-GMP utilized in formation of bacterial bio-films.]] | ||
[[Image:DgcZ all domains sites labeled.png|250 px|left|thumb|'''Figure 4: Diguanylate cyclase DgcZ from ''E. Coli''.''' The domains of the enzyme are labeled, as well as the allosteric binding sites and the Zn<sup>+2</sup> binding sites on the CZB domains<sup>[3]</sup>.]] | [[Image:DgcZ all domains sites labeled.png|250 px|left|thumb|'''Figure 4: Diguanylate cyclase DgcZ from ''E. Coli''.''' The domains of the enzyme are labeled, as well as the allosteric binding sites and the Zn<sup>+2</sup> binding sites on the CZB domains<sup>[3]</sup>.]] | ||
DgcZ is a dimeric protein with | DgcZ is a dimeric protein with two domains per monomer<sup>[4]</sup>. The DgcZ protein has <scene name='69/694239/C2_symmetry/6'>C2</scene> symmetry down its central axis. The catalytic glycine-glycine-glutamate-glutamate-phenylalanine (GGEEF) domains are responsible for synthesizing c-di-GMP. The regulatory chemoreceptor Zinc binding (CZB) domains house the two zinc binding sites, one site located on each monomer. DgcZ binds Zinc in the CZB domains with sub-femtomolar (10<sup>-16</sup>M) affinity. When Zinc is bound, the CZB and GGEEF domains adopt conformations that inhibit DgcZ function, as illustrated in Figure 3<sup>[3]</sup>. Enzyme DgcZ was co-crystallized with Zinc, fixing the structure in its inactivate conformation. The GGEEF domains are catalytic, containing the active sites for cyclizing GTP into c-di-GMP. The CZB domains are used for ligand-mediated regulation of c-di-GMP production and have an important role in signal transduction of bacteria. <scene name='69/694239/Dgcz_ggeef_dom_and_czb_dom/3'>CZB and GGEEF domains</scene> are found in proteins from many bacterial lineages, including DgcZ homologs<sup>[5]</sup>. | ||
==Catalytic GGEEF Domains== | ==Catalytic GGEEF Domains== | ||
The <scene name='69/694239/Ggeef_domain_zmout_dgcz/3'>GGEEF domains</scene> of DgcZ are part of the GGDEF family of proteins that are characterized by a conserved sequence, GG[DE][DE]F<sup>[6]</sup>. The GGEEF domains contain a central five-stranded β-sheet surrounded by five α-helices (Figure 4). The GGEEF domains each possess a catalytic half-site (residues Gly-206, Gly-207, Glu-208, Glu-209, and Phe-210) that, when combined together in a productive conformation, form the entire <scene name='69/694239/Ggeef_domain_dgcz/6'>active site</scene>. Each half-site binds the guanine base of a single GTP molecule via hydrogen bonding to residues <scene name='69/694239/Gtp_guanine_bonds_asn_asp_dgcz/7'>Asn 173 and Asp 182</scene>. This scene depicts an inactive form of the protein because it was crystallized with zinc bound, destroying the active conformation of GGEEF domains. The ribose of each guanosine triphosphate, and subsequent product c-di-GMP riboses, are held only loosely by the enzyme, while the phosphate groups are not bound to the active site at all<sup>[3]</sup>. | The <scene name='69/694239/Ggeef_domain_zmout_dgcz/3'>GGEEF domains</scene> of DgcZ are part of the GGDEF family of proteins that are characterized by a conserved sequence, GG[DE][DE]F<sup>[6]</sup>. The GGEEF domains contain a central five-stranded β-sheet surrounded by five α-helices (Figure 4). The GGEEF domains each possess a <scene name='69/694239/Ggeef_domain_half_site_dgcz/5'>catalytic half-site</scene> (residues Gly-206, Gly-207, Glu-208, Glu-209, and Phe-210) that, when combined together in a productive conformation, form the entire <scene name='69/694239/Ggeef_domain_dgcz/6'>active site</scene>. Each half-site binds the guanine base of a single GTP molecule via hydrogen bonding to residues <scene name='69/694239/Gtp_guanine_bonds_asn_asp_dgcz/7'>Asn 173 and Asp 182</scene>. This scene depicts an inactive form of the protein because it was crystallized with zinc bound, destroying the active conformation of GGEEF domains. The ribose of each guanosine triphosphate, and subsequent product c-di-GMP riboses, are held only loosely by the enzyme, while the phosphate groups are not bound to the active site at all<sup>[3]</sup>. | ||
The alpha phosphate is available for attack by the 3 prime hydroxyl group on another GTP. A <scene name='69/694239/Gtp_magnesium_cofactors_dgcz/3'>Magnesium ion</scene> (Mg<sup>+2</sup>) stabilizes the negative charges on the phosphate groups. When in the inactive conformation, the C3 of the ribose on one GTP is <scene name='69/694239/Gtp_distances/2'>too far away</scene> (9-10 Angstroms) from the alpha phosphate on the other GTP to undergo cyclization. When in the productive conformation, each GTP is held in close proximity with the α-phosphate groups overlapping C3 of the ribose ring. This conformation allows the α-phospate of one GTP to react with the alcohol group attached to C3 of the ribose on the second GTP, resulting in a cyclization of the two molecules into c-di-GMP<sup>[3]</sup>. | The alpha phosphate is available for attack by the 3 prime hydroxyl group on another GTP. A <scene name='69/694239/Gtp_magnesium_cofactors_dgcz/3'>Magnesium ion</scene> (Mg<sup>+2</sup>) stabilizes the negative charges on the phosphate groups. When in the inactive conformation, the C3 of the ribose on one GTP is <scene name='69/694239/Gtp_distances/2'>too far away</scene> (9-10 Angstroms) from the alpha phosphate on the other GTP to undergo cyclization. When in the productive conformation, each GTP is held in close proximity with the α-phosphate groups overlapping C3 of the ribose ring. This conformation allows the α-phospate of one GTP to react with the alcohol group attached to C3 of the ribose on the second GTP, resulting in a cyclization of the two molecules into c-di-GMP<sup>[3]</sup>. | ||
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==CZB Domains and Zinc Binding Site== | ==CZB Domains and Zinc Binding Site== | ||
The <scene name='69/694239/Zb_domain_residues_19-90/7'>CZB domains</scene>, residues 19-90, | The <scene name='69/694239/Zb_domain_residues_19-90/7'>CZB domains</scene>, residues 19-90, regulate the function of DgcZ due to the presence of two <scene name='69/694239/Zinc_binding_domain_zmout/2'>allosteric binding sites</scene>, which bind Zinc cooperatively. 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>[3]</sup>. | ||
[[Image:Zinc binding site labels.jpg|250 px|left|thumb|'''Figure 6: Zn<sup>+2</sup> Coordination to amino acid residues.''' Three of the four 𝝰 helices of the CZB domains of DgcZ coordinate to the Zn<sup>+2</sup> ion for binding.]] | [[Image:Zinc binding site labels.jpg|250 px|left|thumb|'''Figure 6: Zn<sup>+2</sup> Coordination to amino acid residues.''' Three of the four 𝝰 helices of the CZB domains 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 Zinc co-purified with the protein. Zinc allosterically inhibits the activity of enzyme DgcZ through two allosteric binding sites located on the CZB domains <sup>[8]</sup>. The function of the active site of the GGEEF domains are thus inhibited and regulation is prevented. The CZB domains are folded into four anti-parallel α-helices as 2-fold symmetric homodimers, with the N-terminus on helix 𝝰4. The allosteric binding site includes a <scene name='69/694239/Zinc_binding_domain_zm_in/2'>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 each CZB domain and coordinating the Zinc residue in a tetrahedral fashion (Figure 6). For <scene name='69/694239/Ntermctermdgcz/2'>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<sup>[3]</sup>. | 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 Zinc co-purified with the protein. Zinc allosterically inhibits the activity of enzyme DgcZ through two allosteric binding sites located on the CZB domains <sup>[8]</sup>. The function of the active site of the GGEEF domains are thus inhibited and regulation is prevented. The CZB domains are folded into four anti-parallel α-helices as 2-fold symmetric homodimers, with the N-terminus on helix 𝝰4. The allosteric binding site includes a <scene name='69/694239/Zinc_binding_domain_zm_in/2'>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 each CZB domain and coordinating the Zinc residue in a tetrahedral fashion (Figure 6). For <scene name='69/694239/Ntermctermdgcz/2'>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<sup>[3]</sup>. | ||