Sandbox Reserved 1072: Difference between revisions

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OCA, Elizabeth Hughes, Nicole Zimmerman, Geoffrey C. Hoops, David Emch, Isobel Bowles, Jack Trittipo

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 The <scene name='69/694239/Zb_domain_residues_19-90/7'>CZB domains</scene>, residues 19-90, are responsible for regulating the function of DgcZ due to the presence of two Zinc binding sites. The CZB domains contain the <scene name='69/694239/Zinc_binding_domain_zmout/2'>allosteric binding sites</scene> of the enzyme (Figure 4), which exhibit 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>[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.]]
-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 inhibition prevents regulation of GGDEF domain function, the location of the active site. 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>.
 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. When not coordinated to Zn<sup>+2</sup>, the CZB domains presumably adopt a conformation that straightens the <scene name='69/694239/Czbd_with_helices_labeled/4'>α2 helix</scene>. This results in a closer association between <scene name='69/694239/Hydrophobicity_int_residues/5'>hydrophobic residues</scene> on the alpha helices in the already <scene name='69/694239/Hydrophobicity_sf/2'>hydrophobic interior</scene> of the CZB domains. Increased activity of DgcZ presumably occurs due to this conformational shift, which forces the GGEEF domains into a productive conformation. Until the entire protein is crystallized without Zn<sup>+2</sup>, this conformational change is merely hypothesized. But, it is known that activity increases without Zinc due to activation of poly-GlcNAc production, biofilm formation, and maximal cyclic di-GMP production.