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==CzrA==
<StructureSection load='2KJB' size='340' side='right' caption='The dimer CzrA' scene=''>


=Mycolyl Transferase Ag85C=
== Background ==
==Introduction==
[https://en.wikipedia.org/wiki/Operon Operons] are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which 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. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, inhibiting the binding and/or progression of [http://proteopedia.org/wiki/index.php/RNA_polymerase RNA polymerase] to the structural genes, thus inhibiting transcription of the genes into mRNA.  If the regulator protein were to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is 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, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. The Chromosome Determined Zinc Responsible (Czr) operon acts in exactly this manner, Czr A specifically is the regulator protein. The role of Czr A in the Czr operon is described in further detail under biological function.
Antigen 85C is one of three homologous protein components of the Ag85 complex in the cell wall of ''M. tuberculosis''.  This serine esterase enzyme catalyzes the transfer of mycolyl groups, characteristic components of the cell wall of mycobacteria. Several three dimensional structures of Ag85C have been solved, including the wild type enzyme as well as active site variants due to site-directed mutagenesis and covalent modification.
In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn <sup>2+</sup> in the cell.


<StructureSection load='1dqz' size='400' side='right' caption='Antigen 85C in ''Mycobacterium Tuberculosis''' scene=''>
[[Image:operon.png|600px|thumb|center|Visual Overview of Czr Operon]]


==Biological Role==
== Biological Function ==
The unusually thick and waxy cell wall of [http://en.wikipedia.org/wiki/Mycobacterium mycobacteria] is primarily composed of [http://en.wikipedia.org/wiki/Peptidoglycan peptidoglycans], [http://en.wikipedia.org/wiki/Arabinogalactan arbinogalactans], and [http://en.wikipedia.org/wiki/Mycolic_acid mycolic acids]. The mycolic acids, which have very long carbon chains and exhibit extreme hydrophobicity, are responsible for forming the outermost layer of the cell wall, thus creating a hydrophobic envelope surrounding the mycobacterium. Much of the mycolic acid content of the cell wall is in the form of esters (trehalose-6-monomycolate, TMM) and bis-esters (trehalose-6,6'-dimycolate, TDM, cord factor) of trehalose.
Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon <ref>DOI: 10.1073/pnas.0905558106</ref>. The Czr operon contains genes for the proteins Czr A and [http://proteopedia.org/wiki/index.php/3byr CzrB]. Czr B is a Zinc transport protein which moves Zn<sup>2+</sup> out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B<ref>DOI: 10.1073/pnas.0905558106</ref>. Decreased expression of Czr B results in the ability of the cell to retain Zn<sup>2+</sup> more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn<sup>2+</sup> ions<ref>DOI: 10.1073/pnas.0905558106</ref>, which is ideal in that this allows for expression of Czr B, a Zn<sup>2+</sup> transporter to be dependent on the relative amount of Zn<sup>2+</sup> in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn<sup>2+</sup>, in this conformation the <scene name='69/694220/A5_helices__dna_binding/1'>a5 helices are open</scene>. The <scene name='69/694220/A5_helices_dna_binding/1'>a5 helices swing down</scene> to achieve the other conformation which binds two Zn<sup>2+</sup> ions and has relatively low affinity for DNA.
===DNA Binding ===
Czr A performs it's primary function when bound to DNA. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn<sup>2+</sup> inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.
===Zinc Binding ===
Zinc acts as an inhibitor to Czr A, thus preventing transcriptional repression of Czr B and allowing Zn<sup>2+</sup> transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn<sup>2+</sup> transport out of the cell.  


The antigen 85 (Ag85) complex in [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''Mycobacterium tuberculosis''] is composed of three homologous proteins: Ag85A, Ag85B, and Ag85C, encoded by the ''fbpA'', ''fbpB'', and ''fbpC'' genes, respectively. Each of these Ag85 components (A, B, and C) catalyze mycolyl transfer from one molecule of TMM to another (Figure 1) using a catalytic serine triad.  
== Structural Overview ==
NEED REFERENCES!
CzrA functions as a [https://en.wikipedia.org/wiki/Dimer_(chemistry) dimer]. The <scene name='69/694218/Monomeric_unit/1'>monomeric units</scene> dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains <scene name='69/694218/Helices/1'>five alpha helices</scene> seen in purple and <scene name='69/694218/B_sheets/1'>two beta sheets</scene> displayed in yellow. While the function of the [https://en.wikipedia.org/wiki/Beta_sheet beta sheets] are not yet known, key [https://en.wikipedia.org/wiki/Alpha_helix helices] regulate the binding of DNA and Zn<sup> +2 </sup>. The <scene name='69/694218/Alpha_4_helix/1'>alpha 4 helix</scene> is the location of DNA binding and the <scene name='69/694218/Alpha_5_helix/1'>alpha 5 helix</scene> contains the Zn<sup> +2 </sup> binding site. As Zn<sup> +2 </sup> binds, the alpha 4 helices are <scene name='69/694218/Alpha_4_helices_pushed/1'>pushed out of alignment</scene>, repressing their DNA binding ability.
INSERT '''SIMPLE''' REACTION SCHEME! (see Gobec et al.)


===Clinical Relevance===
== Binding of DNA ==
The hydrophobic envelope created by these 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='69/694219/Serandhisresidues/2'>main DNA interactions</scene> have been found to occur at the Ser 54 and 57 along with His 58 residues. These residues 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 R helix. 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>DOI: 10.1073/pnas.0905558106</ref>, the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, 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<sub>a</sub>, 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 fully inhibited Zn<sup>2+</sup> bound state. Table 1 in this same article shows the different K<sub>observed</sub>, and the measured decrease in K<sub>observed</sub> 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. [[Image:Capture01.PNG|300px|center|thumb| Comparison of Val, Ala, and Gln residues]]


==Structure==
The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively<ref>DOI: 10.1021/ja208047b</ref>. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA.
[[Image:Substrate Binding 2D Surface.jpg|200 px|left|thumb|'''Figure 1:''' Octylthioglucoside, a substrate analog, shown in the binding pocket of [http://www.rcsb.org/pdb/explore/explore.do?structureId=1VA5 Ag85C]]]


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 ==
== Zinc Binding ==
 
Most 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<sup> +2</sup>] ions may bind to the dimer<ref>DOI:10.1073/pnas.0636943100</ref>, 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'>swing down</scene> to inhibit the DNA binding residues. 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<sup>+2</sup> cannot bind to the monomer. The <scene name='69/694218/Zinc_residues/1'>zinc binding site</scene> is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. 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>.
[[Image:Substrate_Catalytic_Triad.jpg|300 px|right|thumb|'''Figure 2:''' Relation of the catalytic triad to the octylthioglucoside analog in [http://www.rcsb.org/pdb/explore/explore.do?structureId=1VA5 Ag85C]]]
 
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. 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. This reaction is shown in '''Figure 3''' below.
 
[[Image:Mech_Ag85C.jpeg|400 px|center|thumb|'''Figure 3:''' General reaction catalyzed by Antigen 85C]] 
 
== Methods of Inhibition ==
 
[[Image:C209.jpeg|200 px|left|thumb|'''Figure 4:''' Cys209 stabilizing kinked formation of alpha-9 helix in the native [http://www.rcsb.org/pdb/explore/explore.do?structureId=1dqz Ag85C] enzyme ]]
 
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 <scene name='69/694220/C209/1'>C209 and L232-T 23</scene>, which can be seen in both the scene and '''Figure 4'''. 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|'''Figure 5:''' Ebselen inhibition relaxing the alpha-9 helix in [http://www.rcsb.org/pdb/explore/explore.do?structureId=4qdu Ag85C-Ebselen]]]
 
 
Ag85C can be inhibited by [http://en.wikipedia.org/wiki/Ebselen ebselen], which covalently bounds to the sulfur in C209. Ebselen is a thiol-modifying agent that serves as an electrophile for a C209 nucleophilic attack that results in sulfur-selenium bond formation. 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-T231, which effectively disrupts the interaction that holds the α9 helix in the active conformation. The disruption of this interaction causes the α9 helix to <scene name='69/694220/Inhibited_relaxed_helix/1'>relax</scene>. Furthermore, the bulk of ebselen creates steric hindrance with the α9 helix residues, which can be seen in '''Figure 5'''. The pink helix represents the native enzyme, and the tan helix represents Ag85C covalently bound to ebselen, which is shown in green. Relaxation of the α9 helix due to ebselen removes E228 and H260, which now interacts with S148, from the active site. The absence of these residues destroys the charge relay mechanism, and as a result, the nucleophilicity of the S124 alcohol is not longer strengthened, which decreases serine hydrolytic activity.
 
 
===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|thumb|'''Figure 6:''' Known thiol-reactive inhibitors of Ag85C]]
 
</StructureSection>


Zinc<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 CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn<sup>+2</sup> to bind to CzrA with reasonable affinity and speed in vivo. The zinc<sup>+2</sup> ion forms a tetrahedral complex with the four residues (Figure 1). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized  to bind Zn<sup>+2</sup> with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn<sup>+2</sup> to the dimer will activate the czr operon. Zn<sup>+2</sup> is preferred as CzrB opens a Zn<sup>+2</sup> channel, allowing the excess zinc ions to export the cell.
[[Image:Zinc tetrahedral complex.PNG|thumb|center| Figure 1:Zn<sup>+2</sup> tetrahedral binding complex]]
== References ==
== References ==
<references/>
<references/>

Latest revision as of 20:34, 31 March 2017

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CzrACzrA

<StructureSection load='2KJB' size='340' side='right' caption='The dimer CzrA' scene=>

BackgroundBackground

Operons are a critical genetic component of most prokaryotic cells. There are many different operons that are responsible for the production of proteins with a wide range of functions, the most well-known of which 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. Structurally, each operon contains a regulator, an operator, and one or more structural genes. The regulator protein is responsible for managing the expression level of the structural genes, the operator is similar to a promoter in a regular gene and is where transcription begins, and the structural genes code for proteins. The regulator protein (produced as a result of expression of the regulator gene) most often acts in a repressive manner, though this is not always the case. That is, the regulator protein will bind to the operator of the operon, 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 to consistently be active, there could never be adequate expression of the structural genes, so there must be a way to inactive the regulator protein, thus enabling expression of the structural genes. This is 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, that is the inhibitor is not something that mimics DNA and binds to the active site physically blocking DNA from binding. Rather, the inhibitor of the regulator binds to somewhere other than the active site of the protein, changing it in some way which decreases the proteins affinity or ability to bind DNA. The Chromosome Determined Zinc Responsible (Czr) operon acts in exactly this manner, Czr A specifically is the regulator protein. The role of Czr A in the Czr operon is described in further detail under biological function. In addition to being a component of an operon, Czr A is also considered to be a metal sensor protein. While the immediate function of Czr A is gene regulation, this serves the larger purpose of acting to maintain an appropriate concentration of Zn 2+ in the cell.

Visual Overview of Czr Operon

Biological FunctionBiological Function

Czr A is a transcriptional repressor protein responsible for the regulation of the Czr operon [1]. The Czr operon contains genes for the proteins Czr A and CzrB. Czr B is a Zinc transport protein which moves Zn2+ out of a cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to DNA, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B[2]. Decreased expression of Czr B results in the ability of the cell to retain Zn2+ more readily. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is noncompetitively inhibited by the binding of two Zn2+ ions[3], which is ideal in that this allows for expression of Czr B, a Zn2+ transporter to be dependent on the relative amount of Zn2+ in the cell. Czr A displays two different conformations; the first typically binds DNA and has relatively low affinity for Zn2+, in this conformation the . The to achieve the other conformation which binds two Zn2+ ions and has relatively low affinity for DNA.

DNA BindingDNA Binding

Czr A performs it's primary function when bound to DNA. Each monomeric subunit of the protein binds DNA individually, coming together once attached to the DNA. While bound, Czr A prevents the transcription of the DNA in the Czr operon, acting as a repressor protein and effectively turning off the operon. As was briefly mentioned above, the Czr operon contains the gene responsible for producing Czr B, a metal transport protein which regulates the concentration of zinc in the cell. So, by extension, Czr A is responsible for retaining Zn2+ inside the cell by inhibiting the production of the protein responsible for transporting zinc out of the cell.

Zinc BindingZinc Binding

Zinc acts as an inhibitor to Czr A, thus preventing transcriptional repression of Czr B and allowing Zn2+ transport out of the cell. This allows for zinc transport to essentially be self regulated. That is, when zinc concentration in the cell is relatively high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn2+ transport out of the cell.

Structural OverviewStructural Overview

CzrA functions as a dimer. The dimerize at the czr operon, repressing gene transcription. Each monomeric unit contains seen in purple and displayed in yellow. While the function of the beta sheets are not yet known, key helices regulate the binding of DNA and Zn +2 . The is the location of DNA binding and the contains the Zn +2 binding site. As Zn +2 binds, the alpha 4 helices are , repressing their DNA binding ability.

Binding of DNABinding of DNA

The have been found to occur at the Ser 54 and 57 along with His 58 residues. These residues 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 R helix. The residues involved in the 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 [4], the DNA bound state of CzrA was tested by using the known critical residues for DNA interactions. Critical residues, Gln53, Val42, Ser54, Ser57, and His58, 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 Ka, 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 fully inhibited Zn2+ bound state. Table 1 in this same article shows the different Kobserved, and the measured decrease in Kobserved 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.

Comparison of Val, Ala, and Gln residues

The differences in binding favorability can also be seen when comparing the ΔG for the Apo-state vs. the DNA bound state and the Zinc vs. the Zinc and DNA bound state. These ΔGs were found to be -15.2kcal/mol and -9kcal/mol respectively[5]. This agrees with previously published data showing the Zinc binding inhibits the affinity the protein has to DNA.


Zinc BindingZinc Binding

Most zinc-dependent proteins are transcriptional regulators[6]. CzrA fits into this category as an allosteric inhibitor of the czr operon. Two Zn +2 ions may bind to the dimer[7], at the location of the helix from each monomer. As zinc binds, the alpha 5 helices to inhibit the DNA binding residues. Furthermore, CzrA must be in its dimer form for zinc to bind. The is formed by two residues from each monomer, so Zn+2 cannot bind to the monomer. The is formed by Asp84 and His86 from one monomer, and His97 and His100 from the other monomer. Histidines are a repetitive and commonly found residue in zinc-binding proteins [8].

Zinc+2 binding is driven by a large entropic gain [9]. Water molecules around the metal ion and CzrA protein are displaced, and gain greater freedom. This gain in entropy allows Zn+2 to bind to CzrA with reasonable affinity and speed in vivo. The zinc+2 ion forms a tetrahedral complex with the four residues (Figure 1). This allows other metal ions to act as allosteric inhibitors to CzrA. Any metal that may form a tetrahedral complex will have some affinity for CzrA, assuming it is not too large to fit into the pocket. However, the metal binding pocket of CzrA has been optimized to bind Zn+2 with the highest affinity. As CzrA is a transcriptional repressor, binding of Zn+2 to the dimer will activate the czr operon. Zn+2 is preferred as CzrB opens a Zn+2 channel, allowing the excess zinc ions to export the cell.

Figure 1:Zn+2 tetrahedral binding complex

ReferencesReferences

  1. ↑ DOI: 10.1073/pnas.0905558106
  2. ↑ DOI: 10.1073/pnas.0905558106
  3. ↑ DOI: 10.1073/pnas.0905558106
  4. ↑ DOI: 10.1073/pnas.0905558106
  5. ↑ 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
  6. ↑ 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
  7. ↑ Pennella MA, Shokes JE, Cosper NJ, Scott RA, Giedroc DP. Structural elements of metal selectivity in metal sensor proteins. Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3713-8. Epub 2003 Mar 21. PMID:12651949 doi:http://dx.doi.org/10.1073/pnas.0636943100
  8. ↑ 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.
  9. ↑ 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

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

OCA, Morgan Blake, Sarah Zimmerman, Geoffrey C. Hoops, Jakob Jozwiakowski, Katelyn Baumer
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