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Czr A functions as a [https://en.wikipedia.org/wiki/Protein_dimer dimer] to repress gene transcription. Each <scene name='69/694218/Monomeric_unit/1'>monomeric unit</scene> contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>one antiparallel beta sheet</scene> displayed in yellow. Key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA versus Zn<sup>+2</sup>. The <scene name='69/694220/2kjb_colored_alpha_4/1'>α4 helices</scene> (green) are the location of DNA binding and the <scene name='69/694220/Zinc_pocket_with_residues/2'>α5 helices</scene> (red) contain the Zn<sup>+2</sup> binding sites. | Czr A functions as a [https://en.wikipedia.org/wiki/Protein_dimer dimer] to repress gene transcription. Each <scene name='69/694218/Monomeric_unit/1'>monomeric unit</scene> contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>one antiparallel beta sheet</scene> displayed in yellow. Key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA versus Zn<sup>+2</sup>. The <scene name='69/694220/2kjb_colored_alpha_4/1'>α4 helices</scene> (green) are the location of DNA binding and the <scene name='69/694220/Zinc_pocket_with_residues/2'>α5 helices</scene> (red) contain the Zn<sup>+2</sup> binding sites. | ||
==Allosteric Inhibition by Zn<sup>+2</sup>== | ==Allosteric Inhibition by Zn<sup>+2</sup>== | ||
CzrA is allosterically inhibited by the binding of two Zn<sup>+2</sup> ions. The structure of CzrA has been determined in two different conformations; the first has a high affinity for DNA and has no Zn<sup>+2</sup> ions bound to it (PDB code: 2KJB). In this conformation the <scene name='69/694220/A5_helices__dna_binding/2'>alpha 5 helices are aligned</scene>. Binding of zinc drives a conformational change (PDB code: 2KJC) in which the <scene name='69/694220/A5_helices_dna_binding/2'>alpha 5 helices become unaligned</scene>, changing the overall shape of the protein and significantly lowering its affinity for DNA (Figure 2). Unfortunately, zinc ions are not directly visible in the 2KJC structure, which was determined by NMR spectroscopy. | CzrA is allosterically inhibited by the binding of two Zn<sup>+2</sup> ions. The structure of CzrA has been determined in two different conformations<ref name="critical"/>; the first has a high affinity for DNA and has no Zn<sup>+2</sup> ions bound to it (PDB code: 2KJB). In this conformation the <scene name='69/694220/A5_helices__dna_binding/2'>alpha 5 helices are aligned</scene>. Binding of zinc drives a conformational change (PDB code: 2KJC) in which the <scene name='69/694220/A5_helices_dna_binding/2'>alpha 5 helices become unaligned</scene>, changing the overall shape of the protein and significantly lowering its affinity for DNA (Figure 2). Unfortunately, zinc ions are not directly visible in the 2KJC structure, which was determined by NMR spectroscopy. | ||
[[Image:800px-2KJB + 2KJC side by side.fw.png CROPPED.fw.png|600px|center|thumb| Figure 2: Comparison of Czr A bound to DNA to Czr A with Zn<sup>+2</sup> bound. α5 helices are shown in red and the α4 helices shown in green.]] | [[Image:800px-2KJB + 2KJC side by side.fw.png CROPPED.fw.png|600px|center|thumb| Figure 2: Comparison of Czr A bound to DNA to Czr A with Zn<sup>+2</sup> bound. α5 helices are shown in red and the α4 helices shown in green.]] | ||
== DNA Binding Site== | == DNA Binding Site== | ||
Ser54, Ser57, and His58 are the primary residues involved in <scene name='69/694220/2kjb_colored/3'>DNA interaction</scene> with Czr A <ref name="critical"/>. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA, where the α4 helices (green) <scene name='69/694219/Czra_with_dna/2'>form an interaction with DNA</scene> (Figure 3). Binding of two Zn<sup>+2</sup> ions <scene name='69/694220/Dna_residues_when_inhibited/2'>pushes these residues out of their DNA binding conformation</scene>. Additionally, Val42 and Gln53 (lime green) are involved in the <scene name='69/694220/Val_42_and_gln_53/1'>DNA binding pocket</scene>. | Ser54, Ser57, and His58 are the primary residues involved in <scene name='69/694220/2kjb_colored/3'>DNA interaction</scene> with Czr A<ref name="critical"/>. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA, where the α4 helices (green) <scene name='69/694219/Czra_with_dna/2'>form an interaction with DNA</scene> (Figure 3). Binding of two Zn<sup>+2</sup> ions <scene name='69/694220/Dna_residues_when_inhibited/2'>pushes these residues out of their DNA binding conformation</scene>. Additionally, Val42 and Gln53 (lime green) are involved in the <scene name='69/694220/Val_42_and_gln_53/1'>DNA binding pocket</scene>. | ||
The <scene name='69/694220/Dna_binding_residues/2'>residues directly involved in binding to DNA</scene> Gln53 and Val42 (aqua) as well as the Ser54, Ser57, and His58 (lime) have been individually mutated to Ala, and DNA binding experiments were performed<ref name="critical"/>. Compared to wild type Czr A, Gln53Ala and Val42Ala variants displayed an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Mutations to the main DNA interaction sites Ser54, Ser57, and His58 result in drastic loss of binding similar to the inhibited non-DNA binding conformational state, suggesting that these residues are essential to binding DNA. While the conformational change that occurs from the Zinc bound state to the DNA bound state is small,the α4 helices (shown in green in Figure 2) are slightly shifted. The loss of DNA binding in the mutagenesis experiments in combination with the lack of any other major physical changes between these two states further suggests that the α4 helices are the location of DNA binding in Czr A. A <scene name='69/694220/Czra_with_dna/1'>computational model of CzrA with DNA bound</scene> (not available in the PDB) has been since been published<ref>PMID:22007899</ref> (Figure 3). | The <scene name='69/694220/Dna_binding_residues/2'>residues directly involved in binding to DNA</scene> Gln53 and Val42 (aqua) as well as the Ser54, Ser57, and His58 (lime) have been individually mutated to Ala, and DNA binding experiments were performed<ref name="critical"/>. Compared to wild type Czr A, Gln53Ala and Val42Ala variants displayed an 11-fold and 160-fold decrease in K<sub>a</sub>, respectively. Mutations to the main DNA interaction sites Ser54, Ser57, and His58 result in drastic loss of binding similar to the inhibited non-DNA binding conformational state, suggesting that these residues are essential to binding DNA. While the conformational change that occurs from the Zinc bound state to the DNA bound state is small,the α4 helices (shown in green in Figure 2) are slightly shifted. The loss of DNA binding in the mutagenesis experiments in combination with the lack of any other major physical changes between these two states further suggests that the α4 helices are the location of DNA binding in Czr A. A <scene name='69/694220/Czra_with_dna/1'>computational model of CzrA with DNA bound</scene> (not available in the PDB) has been since been published<ref>PMID:22007899</ref> (Figure 3). | ||
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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]] | ||
Zn<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 Czr A protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to Czr A with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with | Zn<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 Czr A protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to Czr A with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with four residues (Figure 4). Other metal ions that may form a tetrahedral complex will have some affinity for Czr A; however, the metal binding pocket of Czr A has been optimized to bind Zn<sup>+2</sup> with the highest affinity. | ||
</StructureSection> | </StructureSection> | ||