Sandbox Reserved 1053: Difference between revisions

CzrA is allosterically inhibited by the binding of two Zn⁺² ions. The structure of CzrA has been determined in two different conformations; the first has a high affinity for DNA and has no Zn⁺² 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.  

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⁺² 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>.  

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='69/694220/Spacefill_zinc_pockets/1'>zinc binding pockets</scene> are 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. The large number of histidines used in the Czr A zinc pocket is a repetitive and commonly found feature 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>.

Zn⁺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.