Sandbox Reserved 1053: Difference between revisions

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OCA, Ben Zercher, Geoffrey C. Hoops, Katelyn Baumer, Mary Liggett, Jakob Jozwiakowski

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 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 DNA bound state of Czr A was tested by using the known critical residues for DNA interactions . <scene name='69/694220/Dna_binding_residues/2'>residues directly involved in binding to DNA</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 computational model of CzrA with DNA bound (not available in the PDB) has been since been published.
-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, 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 result in binding similar to the inhibited non-DNA binding state, 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 suggests 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:800px-DNABound Final.fw.png CROPPED.fw.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]]