Sandbox Reserved 1070: Difference between revisions

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{{Sandbox_Reserved_Butler_CH462_Sp2015_#}}

= DgcZ from ''E. coli'' =

[[Image:Zn_Binding_Site_DgcZ.png|250 px|left|thumb|Zn Binding Site DgcZ. The ~~Cys~~ reside is not the N-terminal residue, but the rest of the 𝝰helix 2 was not successfully crystallized.]]

[[Image:Zn_Binding_Site_DgcZ.png|250 px|left|thumb|Zn Binding Site DgcZ. The Cys52 reside is not the N-terminal residue, but the rest of the 𝝰helix 2 was not successfully crystallized.]]

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The GGEEF domain includes a central five-stranded β-sheet surrounded by five α-helices. The GGDEF domain includes a conserved sequence of GG[DE][DE]F [2]. Each dimer contains an active half-site that, when combined together in a productive conformation, form the entire active site. Each half-site binds one GTP molecule. The guanyl base forms hydrogen bonds with Asp-173 and Asn-182 to hold it in the active site. A Mg2+ ion stabilizes the negative charges on the phosphate groups. When in the productive conformation, each GTP is held in close proximity with the α-phosphate groups overlapping C3 of the ribose. This conformation allows the α-phospate of one GTP to react with the alcohol group on C3 of the ribose of the other GTP, resulting in a cyclization of the two molecules into c-di-GMP. 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 at all.

=~~==CZB Domain===~~

===Mechanism of Action===

The CZB domain is responsible for regulating the function of DgcZ. Four residues bind zinc with a high affinity even at 10-16M concentrations. Due to the tightness of Zinc binding, the enzyme has not yet been crystallized in the active conformation without the presence of Zinc metal inhibitor.

== Mechanism of Action ==

Diguanylate cyclases only function efficiently as dimers, to bind both GGDEF domains holding the substrates. The presence of Zinc disrupts the ability of the two domains to overlap.

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3. The β and γ phosphates of GTP are kicked off to form c-di-GMP.

== ~~Zinc Ligand(s)~~ ==

===CZB Domain===

~~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~~.

The CZB domain 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 10-16M concentrations. Due to the tightness of Zinc binding, the enzyme has not yet been crystallized in the active conformation without the presence of Zinc metal inhibitor.

[[Image:Zinc coordination DgcZ.png|250 px|left|thumb|Zn Coordination to amino acid residues on three of the four 𝝰 helices of DgcZ]]

=== Zinc Binding Site ===

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 amino acids, H22 of 𝝰1, C52 of 𝝰2, and H79 and H83 of 𝝰3, that span three of the four alpha helices of the CZB domain coordinating the Zinc residue in a tetrahedral fashion. Zahringer et al. mutated Cys52 to Ala, 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 𝝰1 helix shifts, shifting hydrophobic residues 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.

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 amino acids, H22 of 𝝰1, C52 of 𝝰2, and H79 and H83 of 𝝰3, that span three of the four alpha helices of the CZB domain coordinating the Zinc residue in a tetrahedral fashion. Zahringer et al. mutated Cys52 to Ala, 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 𝝰1 helix shifts, shifting hydrophobic residues 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.

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

Sandbox Reserved 1070: Difference between revisions

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@@ Line 1: / Line 1: @@
 {{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE -->
 = DgcZ from ''E. coli'' =
-[[Image:Zn_Binding_Site_DgcZ.png|250 px|left|thumb|Zn Binding Site DgcZ. The Cys reside is not the N-terminal residue, but the rest of the 𝝰helix 2 was not successfully crystallized.]]
+[[Image:Zn_Binding_Site_DgcZ.png|250 px|left|thumb|Zn Binding Site DgcZ. The Cys52 reside is not the N-terminal residue, but the rest of the 𝝰helix 2 was not successfully crystallized.]]
 <Structure load='4h54' size='350' frame='true' align='right' caption='4h54' scene='Insert optional scene name here' />
@@ Line 13: / Line 13: @@
 The GGEEF domain includes a central five-stranded β-sheet surrounded by five α-helices. The GGDEF domain includes a conserved sequence of GG[DE][DE]F [2]. Each dimer contains an active half-site that, when combined together in a productive conformation, form the entire active site. Each half-site binds one GTP molecule. The guanyl base forms hydrogen bonds with Asp-173 and Asn-182 to hold it in the active site. A Mg<sup>2+</sup> ion stabilizes the negative charges on the phosphate groups. When in the productive conformation, each GTP is held in close proximity with the α-phosphate groups overlapping C3 of the ribose. This conformation allows the α-phospate of one GTP to react with the alcohol group on C3 of the ribose of the other GTP, resulting in a cyclization of the two molecules into c-di-GMP. 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 at all.
-===CZB Domain===
+===Mechanism of Action===
-The CZB domain is responsible for regulating the function of DgcZ. Four residues bind zinc with a high affinity even at 10<sup>-16M</sup> concentrations. Due to the tightness of Zinc binding, the enzyme has not yet been crystallized in the active conformation without the presence of Zinc metal inhibitor.
-== Mechanism of Action ==
 Diguanylate cyclases only function efficiently as dimers, to bind both GGDEF domains holding the substrates. The presence of Zinc disrupts the ability of the two domains to overlap.
@@ Line 25: / Line 22: @@
 . The β and γ phosphates of GTP are kicked off to form c-di-GMP.
-== Zinc Ligand(s) ==
+===CZB Domain===
-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.
+The CZB domain 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>-16M</sup> concentrations. Due to the tightness of Zinc binding, the enzyme has not yet been crystallized in the active conformation without the presence of Zinc metal inhibitor.
 [[Image:Zinc coordination DgcZ.png|250 px|left|thumb|Zn Coordination to amino acid residues on three of the four 𝝰 helices of DgcZ]]
 === Zinc Binding Site ===
-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 amino acids, H22 of 𝝰1, C52 of 𝝰2, and H79 and H83 of 𝝰3, that span three of the four alpha helices of the CZB domain coordinating the Zinc residue in a tetrahedral fashion. Zahringer et al. mutated Cys52 to Ala, 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 𝝰1 helix shifts, shifting hydrophobic residues 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.
+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 amino acids, H22 of 𝝰1, C52 of 𝝰2, and H79 and H83 of 𝝰3, that span three of the four alpha helices of the CZB domain coordinating the Zinc residue in a tetrahedral fashion. Zahringer et al. mutated Cys52 to Ala, 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 𝝰1 helix shifts, shifting hydrophobic residues 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.
 This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.