Sandbox Reserved 1053: Difference between revisions
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Ser 54, Ser 57, and His 58 have been found to be the main <scene name='69/694219/Serandhisresidues/3'>sites of DNA interaction</scene>. <scene name='69/694220/2kjb_dna_alpha_4_helix/1'>These residues</scene> are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA, where the alpha 4 helices <scene name='69/694219/Czra_with_dna/2'>form an interaction with DNA</scene>. These residues are found in the N terminal of the alpha 4 helix (figure 3). Additionally, Val 42 and Gln 53 are involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene>. This conclusion was experimentally determined by mutagenesis of the Gln and Val with Ala residues and measuring the mutant DNA binding capacity. In a previously published article <ref name="critical"/>, the DNA bound state of Czr A was tested by using the known critical residues for DNA interactions. <scene name='69/694220/Dna_binding_experiment/1'> Critical DNA binding residues</scene> Gln 53, Val 42 (red), Ser 54, Ser 57, and His 58 (orange) were individually mutated to Ala, and kinetic experiments were performed. Compared to wild type Czr A, mutating Gln53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Mutations to the main DNA interaction sites Ser 54, Ser 57, and His 58 resulted in binding similar to the <scene name='69/694220/Dna_residues_when_inhibited/1'>fully inhibited Zn<sup>2+</sup> bound state</scene>, suggesting that these residues are essential to binding DNA. While the conformational change that occurs from the Zinc to DNA bound state of Czr A is small,the alpha 4 helices (shown in green in Figure 2) are slightly shifted. The loss of DNA binding in the mutagenesis experiements in combination with the lack of any other major physical changes between these two states further supports that the alpha 4 helices are the location of DNA binding in Czr A. Experimental data can be found in table 1 from this same article. | Ser 54, Ser 57, and His 58 have been found to be the main <scene name='69/694219/Serandhisresidues/3'>sites of DNA interaction</scene>. <scene name='69/694220/2kjb_dna_alpha_4_helix/1'>These residues</scene> are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA, where the alpha 4 helices <scene name='69/694219/Czra_with_dna/2'>form an interaction with DNA</scene>. These residues are found in the N terminal of the alpha 4 helix (figure 3). Additionally, Val 42 and Gln 53 are involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene>. This conclusion was experimentally determined by mutagenesis of the Gln and Val with Ala residues and measuring the mutant DNA binding capacity. In a previously published article <ref name="critical"/>, the DNA bound state of Czr A was tested by using the known critical residues for DNA interactions. <scene name='69/694220/Dna_binding_experiment/1'> Critical DNA binding residues</scene> Gln 53, Val 42 (red), Ser 54, Ser 57, and His 58 (orange) were individually mutated to Ala, and kinetic experiments were performed. Compared to wild type Czr A, mutating Gln53 and V42 residues resulted in an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Mutations to the main DNA interaction sites Ser 54, Ser 57, and His 58 resulted in binding similar to the <scene name='69/694220/Dna_residues_when_inhibited/1'>fully inhibited Zn<sup>2+</sup> bound state</scene>, suggesting that these residues are essential to binding DNA. While the conformational change that occurs from the Zinc to DNA bound state of Czr A is small,the alpha 4 helices (shown in green in Figure 2) are slightly shifted. The loss of DNA binding in the mutagenesis experiements in combination with the lack of any other major physical changes between these two states further supports that the alpha 4 helices are the location of DNA binding in Czr A. Experimental data can be found in table 1 from this same article. | ||
[[Image:DNABound Final.PNG|750px|thumb|center| Figure 3: | [[Image:DNABound Final.PNG|750px|thumb|center| Figure 3: Two views of Czr A bound to DNA. A segment of DNA is shown in orange with the alpha 5 helices displayed in red and the alpha 4 helices shown in green]] | ||
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[[Image:Zinc tetrahedral complex.PNG|350px|thumb|center| Figure 4: Zn<sup>+2</sup> tetrahedral binding complex]] | [[Image:Zinc tetrahedral complex.PNG|350px|thumb|center| Figure 4: Zn<sup>+2</sup> tetrahedral binding complex]] | ||
Zinc<sup>+2</sup> binding is driven by a large [https://en.wikipedia.org/wiki/Entropy entropic] gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 4) | Zinc<sup>+2</sup> binding is driven by a large [https://en.wikipedia.org/wiki/Entropy entropic] gain <ref>DOI:10.1021/ja906131b</ref>. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 4), allowing other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell. | ||
</StructureSection> | </StructureSection> | ||
