Sandbox Reserved 1053: Difference between revisions

Ser 54, Ser 57, and His 58 are the primary sites of <scene name='69/694220/2kjb_colored/3'>DNA interaction</scene> in Czr A. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA, where the alpha 4 helices (green) <scene name='69/694219/Czra_with_dna/2'>form an interaction with DNA</scene>. Additionally, Val 42 and Gln 53 (lime green) are involved in the <scene name='69/694220/Val_42_gly_53/3'>DNA binding pocket</scene>. This conclusion was experimentally determined by mutagenesis of the Gln and Val residues with an Ala and measuring the mutant DNA binding capacity. The DNA bound state of Czr A was tested by using the known critical residues for DNA interactions <ref name="critical"/>. <scene name='69/694220/Dna_binding_residues/1'>Critical DNA binding residues</scene> Gln 53, Val 42 (aqua), Ser 54, Ser 57, and His 58 (lime) 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_a, 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/2'>inhibited Zinc 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 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.  

Many zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. Czr A fits into this category as an [https://en.wikipedia.org/wiki/Allosteric_regulation allosteric inhibitor] of the czr operon. Two [https://en.wikipedia.org/wiki/Zinc Zn⁺²] ions may bind to the dimer<ref name="critical"/>, at the location of the <scene name='69/694220/A5_helices__zn_binding/2'>alpha 5 helix</scene> from each monomer. As zinc binds, the alpha 5 helices <scene name='69/694218/2kjc_zinc_bound/1'>unalign</scene> to inhibit the DNA binding residues (Figure 2). Furthermore, CzrA must be in its dimer form for zinc to bind. The <scene name='69/694218/Spacefill_with_zinc_pockets/1'>zinc binding pocket</scene> is formed by two residues from each monomer, so Zn⁺² cannot bind to the monomer. The <scene name='69/694220/Zinc_binding_residues/7'>zinc binding site</scene> is formed by Asp 84 and His 86 from one monomer, and His 97 and His 100 from the other monomer. Zinc ions were not present in the solution NMR structure, so a representation of a zinc ion in the binding pocket can be seen in figure 4. Histidine residues are a repetitive and commonly found residue in zinc-binding proteins <ref>Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun 4;4(6):1609-1614.</ref>.

Zinc⁺² 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⁺² to bind to Czr A with reasonable affinity and speed in vivo. The zinc⁺² ion forms a tetrahedral complex with the four residues (Figure 4), allowing other metal ions to also act as allosteric inhibitors to Czr A. Any metal that may form a tetrahedral complex will have some affinity for Czr A, assuming it is not too large to fit into the pocket. However, the metal binding pocket of Czr A has been optimized  to bind Zn⁺² with the highest affinity. As Czr A is a transcriptional repressor, binding of Zn⁺² to the dimer will activate the czr operon. Zn⁺² is preferred as Czr B opens a Zn⁺² channel, allowing the excess zinc ions to export the cell.