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=Zinc Dependent Transcriptional Repressor of the Czr operon (CzrA)=
== Background ==
<StructureSection load='CzrAwithDNA.pdb' size='340' frame='true' side='right' caption='The dimer Czr A' scene=''>
<scene name='69/694220/Czra_with_dna/1'>CzrA</scene> 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''.
==Biological Function==
===Operon Overview===
[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:Operon.png|500px|thumb|center|Figure 1: Overview of CzrA operon structure]]
===The Czr Operon===
The <u>C</u>hromosome determined <u>z</u>inc <u>r</u>esponsible (Czr) operon acts as described above (Figure 1), with CzrA acting as a regulator protein to the downstream structural gene CzrB<ref name="critical">Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a
paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43
18177-18182.</ref>. The CzrB gene in turn codes for a Zn<sup>+2</sup> pump, the [http://proteopedia.org/wiki/index.php/3byr 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<sup>+2</sup> inside the cell, as there are fewer pumps to export Zn<sup>+2</sup>. This metal sensing system serves to maintain an appropriate intracellular concentration of Zn<sup>+2</sup>. 
==Structural Overview==
Czr A functions as a [https://en.wikipedia.org/wiki/Protein_dimer dimer] to repress gene transcription.  Each <scene name='69/694218/Monomeric_unit/1'>monomeric unit</scene> contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>one antiparallel beta sheet</scene> displayed in yellow. Key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA versus Zn<sup>+2</sup>. The <scene name='69/694220/2kjb_colored_alpha_4/1'>α4 helices</scene> (green) are the location of DNA binding and the <scene name='69/694220/Zinc_pocket_with_residues/2'>α5 helices</scene> (red) contain the Zn<sup>+2</sup> binding sites.
==Allosteric Inhibition by Zn<sup>+2</sup>==
CzrA is allosterically inhibited by the binding of two Zn<sup>+2</sup> ions. The structure of CzrA has been determined in two different conformations<ref name="critical"/>; the first has a high affinity for DNA and has no Zn<sup>+2</sup> 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.
[[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<sup>+2</sup> bound. α5 helices are shown in red and the α4 helices shown in green.]]
== DNA Binding Site==
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<sup>+2</sup> 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 antigen 85 (ag85) complex in [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''Mycobacterium tuberculosis''], which is responsible for causing the disease [http://en.wikipedia.org/wiki/Tuberculosis Tuberculosis], is composed of three intracellular membrane proteins: Ag85A, B, and C. The Ag85 complex is a major component of the cell wall, with each protein catalyzing the transfer of important cell wall constituents into the membrane. <ref>PMID: 10655617</ref> The cell wall of ''Mycobacterium tuberculosis'' is composed of three primary molecules: [http://en.wikipedia.org/wiki/Peptidoglycan peptidoglycans], [http://en.wikipedia.org/wiki/Arabinogalactan arbinogalactans], and [http://en.wikipedia.org/wiki/Mycolic_acid mycolic acids]. Ag85C is of particular interest due to its transfer of mycolic acids, which is one of the major components in determining cell wall integrity. The mycolic acids are responsible for forming the outermost layer of the cell wall. Mycolic acids have a long fatty acid chain and exhibit extreme hydrophobicity, which effectively creates a hydrophobic envelope surrounding the bacterium. The hydrophobic envelope created by they mycolic acids creates a barrier against small hydrophilic molecules, such as Tuberculosis antibiotics. By targeting this mycoloyltransferase activity, inhibition of Ag85C offers potential for cell wall disruption and subsequent antibiotic targeting for normally drug-resistant ''Mycotaberia tuberculosis''. <ref>PMID: 10200974</ref>
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<sub>a</sub>, 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).
==Structure==
<StructureSection load='1dqz' size='400' side='right' caption='Antigen 85C in ''Mycobacterium Tuberculosis''' scene=''>


== General Structure ==
[[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.]]
 
[[Image:Substrate Binding 2D Surface.jpg|200 px|left|thumb|Octylthioglucoside, a substrate analog, shown in the binding pocket of Antigen 85C]]
== Zinc Binding Site==
 
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<sup> +2</sup>] 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<sup>+2</sup> 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>.
Antigen 85C was crystallized in its dimeric form.<ref>PMID: 25028518</ref> The <scene name='69/694220/Secondary_structures/2'>secondary structure</scene> shown in the monomeric form is composed of helices, shown in pink, with one interwoven beta sheet, shown in yellow. The confrontation of a central β-sheet bordered by α–helices creates an a α/β hydrolase fold in Ag85C, and this tertiary conformation is highly conserved across enzymes that function in this capacity. <ref>PMID:10655617</ref> The substrate binding pocket of Ag85C is composed of two separate but equally important components; there is carbohydrate binding pocket for the trehalose, and there is a fatty acid binding pocket for the mycolic acid. As a result, trehalose monomycolate can effectively bind to the Ag85C binding pocket. This binding pocket is shown in the left image with a substrate mimic.
 
== Enzymatic Activity ==
 
Mutagenesis studies have confirmed the Ag85C functions through a Glu-His-Ser <scene name='69/694220/Catalytic_triad/4'>catalytic triad</scene>, similar to that of [http://en.wikipedia.org/wiki/Chymotrypsin chymotrypsin]. By modifying each of the catalytic residues separately testing the enzyme’s relative activity, it has been shown that mutation of any one of these residues dramatically reduces activity (Figure #). The S124 alcohol’s nucleophilicity is inductively strengthened through H260 and E224, which allows the S124 residue to catalyze a reaction that involves [http://en.wikipedia.org/wiki/Cord_factor trehalose 6, 6’-dimycolate]. The formation of the functional catalytic triad relies on upon Van der Waals interaction between C209 and the peptide bond between L232 and T231. This interaction results in a kinked conformation of the α9 helix, which promotes that activity of the catalytic triad. As a result, Ag85C, a mycolyl transferase, can facilitate the modification of trehalose monomycolates to trehalose dimycolates, which are then transported to the bacterial cell wall.
 
[[Image:General_mechanism.jpg|400 px|center|General reaction catalyzed by Antigen 85C]] 


== Methods of Inhibition ==
[[Image:Zinc tetrahedral complex.PNG|350px|thumb|center| Figure 4: Zn<sup>+2</sup> tetrahedral binding complex]]
 
[[Image:C209.jpeg|200 px|left|thumb|Cys209 stabilizing kinked formation of alpha-9 helix]]
 
Due to the importance of Ag85C enzymatic activity in maintaining the integrity of the ''Mycobacteria tuberculosis'' cell wall though mycolic acid modifications, the Ag85C enzyme represents a potentially effective avenue for inhibiting cell growth. The conformational sensitivity of the active site residues, H260, E228, and S124, relies entirely upon Van der Waals interaction between C209 and L232-T 231 (Figure #). The C209 facilitated interaction causes the <scene name='69/694220/Alpha_9_helix/2'>α9 helix</scene> to acquire a kinked conformation that promotes optimal interaction distances between catalytic residues. As a result, C209 has been a specific target residue for Ag85C inhibition.
 
 
[[Image:Ebselen_inhibition.jpeg|200 px|right|thumb|Ebselen inhibition relaxing the alpha-9 helix]]
 
 
Ag85C can be inhibited by [http://en.wikipedia.org/wiki/Ebselen ebselen] covalently bound to the sulfur of C209. Ebselen is a thiol-modifying agent that serves as an electrophile for the C209 that results in a sulfur-selenium bond. Co-crystallization of ebselen with Ag85C provides an explanation for the mechanism of ebselen-based inhibition. The addition of ebselen increases the distance between C209 and L232-T31, which effectively disrupts the interaction that holds the α9 helix in the active conformation. Furthermore, the bulk of ebselen creates steric hindrance with the α9 helix residues (Figure #). Relaxation of the α9 helix removes E228 and H260, which now interacts with S148, from the active site. The absence of these residues decreases the nucleophilicity of the S124 alcohol which decreases serine hydrolytic activity.
 
Relaxed alpha helix scene <scene name='69/694220/Inhibited_relaxed_helix/1'>relaxed</scene>
 
===Inhibitors===
 
Additional thiol-modifying agents, [http://en.wikipedia.org/wiki/4-Chloromercuribenzoic_acid p-chloromercuribenzoic acid] and [http://en.wikipedia.org/wiki/Iodoacetamide iodoacetamide], were crystalized with Ag85C. The structures show that each of these thiol-reactive inhibitors covalently bound to C209 and caused a relaxation of the α9 helix in a similar fashion to ebselen.
   
   
[[Image:Inhibitors_Ag85c.jpeg|400 px|center]]
Zn<sup>+2</sup> 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<sup>+2</sup> to bind to Czr A with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> 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<sup>+2</sup> with the highest affinity.
</StructureSection>


</StructureSection>
__NOTOC__


== References ==
== References ==
<references/>
<references/>
==Student Contributors==
*Katelyn Baumer
*Jakob Jozwiakowski
*Catie Liggett

Latest revision as of 04:40, 19 January 2018

Zinc Dependent Transcriptional Repressor of the Czr operon (CzrA)Zinc Dependent Transcriptional Repressor of the Czr operon (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.

Biological Function

Operon Overview

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 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.

Figure 1: Overview of CzrA operon structure

The Czr Operon

The 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 Overview

Czr A functions as a dimer to repress gene transcription. Each contains seen in purple and displayed in yellow. Key helices regulate the binding of DNA versus Zn+2. The (green) are the location of DNA binding and the (red) contain the Zn+2 binding sites.

Allosteric Inhibition by Zn+2

CzrA is allosterically inhibited by the binding of two Zn+2 ions. The structure of CzrA has been determined in two different conformations[1]; the first has a high affinity for DNA and has no Zn+2 ions bound to it (PDB code: 2KJB). In this conformation the . Binding of zinc drives a conformational change (PDB code: 2KJC) in which the , 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.

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.

DNA Binding Site

Ser54, Ser57, and His58 are the primary residues involved in 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) (Figure 3). Binding of two Zn+2 ions . Additionally, Val42 and Gln53 (lime green) are involved in the .

The 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 (not available in the PDB) has been since been published[2] (Figure 3).

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.

Zinc Binding Site

Many 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 from each monomer. As zinc binds, the α5 helices to inhibit the DNA binding residues (Figure 2). Furthermore, CzrA must be in its dimer form for zinc to bind. The are formed by two residues from each monomer, so Zn+2 cannot bind to the monomer. The 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[1], 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 [4].

Figure 4: Zn+2 tetrahedral binding complex

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). 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.

The dimer Czr A

Drag the structure with the mouse to rotate


ReferencesReferences

  1. ↑ 1.0 1.1 1.2 1.3 1.4 1.5 Arunkumar A., Campanello G., Giedroc D. (2009). Solution Structure of a paradigm ArsR family zinc sensor in the DNA-bound state. PNAS 106:43 18177-18182.
  2. ↑ Chakravorty DK, Wang B, Lee CW, Giedroc DP, Merz KM Jr. Simulations of allosteric motions in the zinc sensor CzrA. J Am Chem Soc. 2012 Feb 22;134(7):3367-76. doi: 10.1021/ja208047b. Epub 2011 Nov , 14. PMID:22007899 doi:http://dx.doi.org/10.1021/ja208047b
  3. ↑ MacPherson S, Larochelle M, Turcotte B. A fungal family of transcriptional regulators: the zinc cluster proteins. Microbiol Mol Biol Rev. 2006 Sep;70(3):583-604. PMID:16959962 doi:http://dx.doi.org/10.1128/MMBR.00015-06
  4. ↑ 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.
  5. ↑ Grossoehme NE, Giedroc DP. Energetics of allosteric negative coupling in the zinc sensor S. aureus CzrA. J Am Chem Soc. 2009 Dec 16;131(49):17860-70. doi: 10.1021/ja906131b. PMID:19995076 doi:http://dx.doi.org/10.1021/ja906131b


Student ContributorsStudent Contributors

  • Katelyn Baumer
  • Jakob Jozwiakowski
  • Catie Liggett

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA, Ben Zercher, Geoffrey C. Hoops, Katelyn Baumer, Mary Liggett, Jakob Jozwiakowski
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