Sandbox Reserved 1053
Zinc Dependent Transcriptional Repressor of the Czr operon (CzrA)
CzrA is a transcriptional repressor protein responsible for the regulation of the Czr operon in prokaryotes. The best studied example to date comes from Staphylococcus aureus. ContentsBiological FunctionOperon OverviewOperons are a critical genetic component of most prokaryotic cells. There are many different operons, responsible for the production of proteins with a wide range of functions. The most well-known and studied operons are the Lac and Trp operons, responsible for producing enzymes which metabolize lactose and tryptophan respectively. Despite many differences in each operon and the proteins that they encode, operons all function in the same general manner (Figure 1). Each operon contains a regulator, an operator, and one or more structural genes. The regulator gene codes for a protein responsible for managing the expression level of the structural genes. The operator contains the binding sequence for RNA polymerase and is the site where transcription begins. Lastly, the structural genes code for proteins to be used elsewhere. The regulator protein (produced as a result of expression of the regulator gene) usually acts in a repressive manner. The regulator protein will bind to the operator gene, inhibiting the binding and/or progression of RNA polymerase to the structural genes, thus inhibiting transcription of the genes into mRNA. If the regulator protein were always active, the structural genes would never be expressed, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is usually achieved through the binding of an inhibitor to the regulator protein. Since regulator proteins are DNA binding proteins, often this inhibition is allosteric rather than competitive. The inhibitor of the regulator protein binds to somewhere other than the active site of the protein, changing the conformation of the regulator protein to decrease its ability to bind DNA and repress transcription.  The Czr OperonThe Chromosome determined zinc responsible (Czr) operon acts as described above (Figure 1), with CzrA acting as a regulator protein to the downstream structural gene CzrB[1]. The CzrB gene in turn codes for a Zn+2 pump, the CzrB protein. When relatively low amounts of zinc are present in the cell CzrA will bind to the operator on the Czr operon, preventing the progression of RNA polymerase and thus inhibiting expression of CzrB. Decreased expression of CzrB results in a buildup of Zn+2 inside the cell, as there are fewer pumps to export Zn+2. This metal sensing system serves to maintain an appropriate intracellular concentration of Zn+2. Structural OverviewCzr A functions as a dimer to repress gene transcription. Each monomeric unit contains five alpha helices seen in purple and one antiparallel beta sheet displayed in yellow. Key helices regulate the binding of DNA versus Zn+2. The α4 helices (green) are the location of DNA binding and the α5 helices (red) contain the Zn+2 binding sites. Allosteric Inhibition by Zn+2CzrA 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 alpha 5 helices are aligned. Binding of zinc drives a conformational change (PDB code: 2KJC) in which the alpha 5 helices become unaligned, 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.  DNA Binding SiteSer54, Ser57, and His58 are the primary residues involved in DNA interaction with Czr A [1]. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA, where the α4 helices (green) form an interaction with DNA (Figure 3). Binding of two Zn+2 ions pushes these residues out of their DNA binding conformation. Additionally, Val42 and Gln53 (lime green) are involved in the DNA binding pocket. The residues directly involved in binding to DNA 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[1]. Compared to wild type Czr A, Gln53Ala and Val42Ala variants displayed an 11-fold and 160-fold decrease in Ka, 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[2] (Figure 3).  Zinc Binding SiteMany zinc-dependent proteins are transcriptional regulators[3]. Czr A fits into this category as an allosteric inhibitor of the czr operon. Two Zn +2 ions may bind to the dimer[1], at the location of the alpha 5 helix from each monomer. As zinc binds, the alpha 5 helices unalign to inhibit the DNA binding residues (Figure 2). Furthermore, CzrA must be in its dimer form for zinc to bind. The zinc binding pockets are formed by two residues from each monomer, so Zn+2 cannot bind to the monomer. The zinc binding site 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 [4].  Zn+2 binding is driven by a large entropic gain [5]. 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.
| ||||||||||||
