Sandbox Reserved 1053: Difference between revisions

The <scene name='69/694219/Serandhisresidues/3'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58 residues. <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. These residues are found in the N terminal of the alpha 4 helix (figure 3). The residues involved in the <scene name='69/694219/Dna_binding_pocket/1'>DNA binding pocket</scene> are Val 42 and Gln 53. This was experimentally determined by mutating the Gln and Val with Ala residues and measuring the binding capacity; In a previously published article <ref name="critical"/>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. <scene name='69/694220/Dna_binding_experiment/1'>Critical DNA binding residues</scene>, Gln53, Val42 (both shown in red), Ser54, Ser57, and His58 (all shown in orange), were replaced with Ala and then compared to the kinetics of the wild type protein. Replacing only the Q53 and V42 residues resulted in an 11-fold and 160-fold decrease in K_a, respectively. Other residues such as S54, S57, and H58 were also replaced with Ala residues, and it was found that these mutations caused binding similar to the <scene name='69/694220/Dna_residues_when_inhibited/1'>fully inhibited Zn²⁺ bound state</scene>. The conformational change that occurs from the Zinc to DNA bound state regarding these residues is small, but the alpha 4 helix (shown in green in Figure 2) does subtly move. Because no major physical change occurs between these two states, it further supports that this region is the main DNA interaction site because of the loss of affinity after the mutation took place. Table 1 in this same article shows the different K_observed, and the measured decrease in K_observed for each mutation. The bind between the DNA and the protein can be attributed to losing certain intermolecular forces such as possible hydrogen bonding when changing from Gln and Ala, and a loss of London Dispersion forces in the Val to Ala change.

Many zinc-dependent proteins are transcriptional regulators<ref>DOI: 10.1128/MMBR.00015-06</ref>. CzrA 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='69/694218/Alpha_5_helices/2'> alpha 5 </scene> helix 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/6'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Zinc ions were not present in the solution NMR crystal structure, so a representation of a zinc ion in the binding pocket can be seen in figure 4. Histidines 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>.