Sandbox Reserved 1053: Difference between revisions

<StructureSection load='CzrAwithDNA.pdb' size='340' frame='true' side='right' caption='The dimer Czr A' scene=''>

[https://en.wikipedia.org/wiki/Operon Operons] 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 [https://en.wikipedia.org/wiki/Lac_operon Lac] and [https://en.wikipedia.org/wiki/Trp_operon 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 [https://en.wikipedia.org/wiki/Regulator_gene regulator], an [https://en.wikipedia.org/wiki/Operator_(biology) operator], and one or more [https://en.wikipedia.org/wiki/Structural_gene 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 [https://en.wikipedia.org/wiki/RNA_polymerase RNA polymerase] and is the site where [https://en.wikipedia.org/wiki/Transcription_(biology) 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 [https://en.wikipedia.org/wiki/Allosteric_regulation 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.

[[Image:800px-2KJB + 2KJC side by side.fw.png CROPPED.fw.png|600px|center|thumb| Figure 2: Comparison of Czr A bound to DNA to Czr A with Zn⁺² bound. α5 helices are shown in red and the α4 helices shown in green.]]

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>.

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_a, 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 <scene name='69/694220/Czra_with_dna/1'>computational model of CzrA with DNA bound</scene> (not available in the PDB) has been since been published<ref>PMID:22007899</ref> (Figure 3).

[[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 α5 helices displayed in red and the α4 helices shown in green.]]

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'>α5 helix</scene> from each monomer. As zinc binds, the α5 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 Asp84 and His 86 from one monomer, as well as His97 and His100 from the other monomer. Zinc ions were not present in the solution NMR structure<ref name="critical"/>, so a representation of a zinc ion in the binding pocket has been drawn 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>.

[[Image:Zinc tetrahedral complex.PNG|350px|thumb|center| Figure 4: Zn⁺² tetrahedral binding complex]]

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).  Other metal ions that may form a tetrahedral complex will have some affinity for Czr A; however, the metal binding pocket of Czr A has been optimized to bind Zn⁺² with the highest affinity.