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| ==Ag85C of ''Mycobacterium tuberculosis''== | | {{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --> |
| <StructureSection load='1dqz' size='340' side='right' caption='Caption for this structure' scene=''> | | ==Your Protein Name here== |
| | <StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''> |
| | This is a default text for your page ''''''. Click above on '''edit this page''' to modify. Be careful with the < and > signs. |
| | You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue. |
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| == '''Introduction''' == | | == Biological Function == |
| [http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''Mycobacterium tuberculosis''] is the bacteria that causes the [http://www.mayoclinic.org/diseases-conditions/tuberculosis/basics/definition/con-20021761 tuberculosis] (TB) disease itself, a leading infectious cause of death world-wide. Thus, it is necessary to develop antimycobacterial drugs. However, this has proven to be difficult as the bacteria can become resistant as a result of misuse of drugs. This gives the bacteria time to mutate which ultimately leads to the drug resistance. To combat this, scientists and researchers have taken on many studies and clinical trials to locate specific drug targets. Obstacles for controlling TB infection include lengthy treatment regimens, drug resistance, lack of a highly efficacious vaccine, and incomplete understanding of the factors that control virulence and disease progression.<ref> Online Mendelian Inheritance in Man (OMIM). Mycobacterium tuberculosis, susceptibility to. [Internet]. Baltimore (MD): Johns Hopkins University; 2014 Nov 12 [cited 2015 March 16]. Available from http://www.omim.org/entry/607948 </ref> Of these projects, some deal specifically with the ''M. tuberculosis'' [http://onlinelibrary.wiley.com/doi/10.1046/j.1472-765x.2002.01091.x/epdf Antigen 85] (Ag85) complex. Ag85 complex consists of 3 secreted enzymes (A, B, and C) and plays a key role in pathogenesis and cell wall synthesis of ''M. tuberculosis''. <ref name="Favrot"> PMID:25028518 </ref>
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| The [http://www.hindawi.com/journals/jir/2011/405310.fig.001.jpg cell wall of mycobacterium tuberculosis] covalently linked molecules, one of which is [https://en.wikipedia.org/wiki/Mycolic_acid mycolic acid], a fatty acid which exists in the membrane as the free glycolipids trehalose monomycolate (TMM) and [http://en.wikipedia.org/wiki/Trehalose_dimycolate trehalose dimycolate] (TDM). Studies suggest that these three components are necessary to maintain the integrity of the cell wall.<ref name="Gobec">PMID:15177473</ref> More specifically, Ag85C is important in the generation of the bacterial cell wall in that it transfers the necessary mycolic acid to the rest of the Ag85 complex, and thus it is suggested that the enzyme displays mycolyltransferace activity. Specifically, the Ag85 enzymes catalyze the transfer of a mycolyl residue from one molecule of α, α-trehalose monomycolate (TMM) to another TMM, leading to the [http://biology.kenyon.edu/BMB/Jmol2009/KatandSuzanne/TDM-Formation.jpg formation of TDM]. Trehalose is necessary for proper growth of the bacterial cells. Thus, there is potential to target these enzymes with specific drugs to prevent the spread of the disease.<ref name="Favrot"/>
| | == Structural Overview == |
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| | == Mechanism of Action == |
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| == '''Clinical Relevance''' == | | == Zinc Ligand(s) == |
| Because the cell wall envelope of ''M. tuberculosis'' is essential for the bacteria’s viability and virulence, it is the primary target for antimycobacterial drugs. More specifically, it is known that the Ag85 complex with its three protein components (A, B, and C) plays a key role in cell wall biosynthesis and the [http://www.nature.com/nchembio/journal/v7/n4/images_article/nchembio.539-F1.jpg transfer of mycolic acid from one molecule of TMM to another]. The resultant molecule, TDM, has been suggested to be important in maintaining ''M. tuberculosis'' cell wall integrity. Studies have also suggested that removal of Ag85C from a strain of ''M. tuberculosis'' results in a significant decrease in the presence of cell-wall linked mycolic acids. Thus, scientists hypothesize that inhibition of Ag85C and its mycolyltransferase activity will disrupt TDM and its ability to maintain cell wall integrity.<ref name="Gobec"/> Knowing this, researchers have suggested that inhibition of mycolic acid transfer and, ultimately, Ag85C, must be involved in the continuously-developing class of antimycobacterial drugs aimed at combatting ''M. tuberculosis''.<ref>PMID:9162010</ref>
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| | == Other Ligands == |
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| == '''Structure''' ==
| | This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes. |
| [[Image:AG85C homodimer.jpg |100 xp|left|thumb|'''Figure 1.''' Ag85C homodimer. ]]
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| Ag85 C is a <scene name='69/694219/Dimer/1'>dimer</scene> of identical subunits (Figure 1), and quite a bit about the amino acid sequence and overall structure of Ag85C is known. The enzyme contains a [http://en.wikipedia.org/wiki/Catalytic_triad catalytic triad] comprised of [http://en.wikipedia.org/wiki/Serine serine], [http://en.wikipedia.org/wiki/Glutamic_acid glutamate], and [http://en.wikipedia.org/wiki/Histidine histidine].
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| This would lead us to think that we could inhibit mycolyl transfer and shut down the enzyme by directly inhibiting the serine residue of the <scene name='69/694218/Catalytic_triad/2'>catalytic triad</scene>, but we can instead modify a near by [http://en.wikipedia.org/wiki/Cysteine cysteine] residue and have the same effect. This was determined by a series of experiments in which scientists generated various mutants and modifications of the Ag85C enzyme.<ref> Research Collaboratory for Structural Bioinformatics Protein Database (RCSB PDB) Diacylglycerol acyltransferase/mycolyltransferase Ag85C - P9WQN9 (A85C_MYCTU). [Internet] San Diego (CA): University of San Diego; 2014 [cited 2015 March 16]. [http://dx.doi.org/10.2210/pdbp9wqn9/pdb DOI:10.2210/pdbp9wqn9/pdb]</ref>
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| ===Active Site===
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| [[Image:AG85C active site 3.jpg |100 xp|left|thumb|'''Figure 2.''' Ag85C with labeled active site residues. The three catalytic residues, Ser124, Glu228, and His260, as well as Cys209 are labeled.]] Within the <scene name='69/697503/Active_site/2'>Ag85C active site</scene>,the homodimers shown in blue and green and the active site shown in white, three residues function together to make up the <scene name='69/694219/Cattri/1'>catalytic triad</scene> for this enzyme (Figure 2). The goal of the catalytic triad is to generate a nucleophilic residue for covalent catalysis by using an acid-base-nucleophile triad. These three residues, Ser124, Glu228, and His260 form a charge-relay network to polarize and activate the nucleophile, Ser124, which is then able to attack the substrate to form a covalent intermediate, which is then hydrolysed to regenerate a free enzyme. This charge relay is an example of the well known [http://en.wikipedia.org/wiki/Chymotrypsin chymotrypsin mechanism]. Overall, it is suggested that increased enzymatic activity is attributed to the components of the active site remaining intact so that the serine nucleophile can react to form an intermediary and stabilize the transition state formed during catalysis. In the native structure, the <scene name='69/694219/Helix/2'>α-helix 9</scene> maintains a kinked conformation necessary for correct formation of the hydrogen bonding network between the residues of the catalytic triad, thus allowing for high enzymatic activity.<ref name="Favrot"/>
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| ===Cysteine 209===
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| <scene name='69/694218/Cysteine_209/2'>Cysteine 209</scene> is located near the <scene name='69/697503/Active_site/2'>Ag85C active site</scene>, but is not directly involved in the enzyme mechanism. Recent studies suggest that this residue plays a significant role in the deactivation of Ag85C, using a mechanism not previously considered for these enzymes. Any mutation or modification to the Cys209 residue results in the relaxation of the otherwise kicked α-helix 9, which disrupts the hydrogen bonding network within the catalytic triad of the enzyme and inactivates Ag85C.<ref name="Favrot"/>
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| =='''Mutations and Modifications'''==
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| Researchers hold particular interest in specific geometric restraints caused by ligands introduced in various Ag85C mutants and modifications: <scene name='69/694218/Ag85c-ebselen/1'>Ag85C-Ebselen</scene>, <scene name='69/694218/Ag85c-hg/1'>Ag85C-Hg</scene>, <scene name='69/694218/Ag85c-e228q/1'>Ag85C-E228Q</scene>, and <scene name='69/694218/Mutationh260q/1'>Ag85C-H260Q</scene>. Other mutants and modifications not described here include [http://proteopedia.org/wiki/index.php/4qdt Ag85C-IAA], [http://proteopedia.org/wiki/index.php/4qdx Ag85C-C209G], and [http://proteopedia.org/wiki/index.php/4qek Ag85C-S124A]. The results demonstrate that modification of Ag85C by [http://en.wikipedia.org/wiki/Thiol thiol]-reactive compounds imparts structural changes in the enzyme’s active site. Specifically, scientists note a disruption in the natural kink in helix α-9. The resulting relaxed conformation of the helix significantly reduces or completely eliminates the enzymatic function of Ag85C.<ref name="Favrot"/>
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| ===Ag85C-ebselen===
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| [[Image:Ag85 ebselen.jpg |100 xp|left|thumb|'''Figure 3.''' [http://proteopedia.org/wiki/index.php/4qdu Ag85C-ebselen]: Ebselen covalently binds to Cys209 and forces the otherwise kinked helix α-9 to take on a relaxed conformation, thus allowing movement of the helix and a reduction in enzymatic activity. The native structure is shown in blue while the modified version is shown in purple, demonstrating a relaxed helix. Ebselen-bound Cys209 is shown in green.]]
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| [[Image:ebselen pic.jpg |100 xp|left|thumb|'''Figure 4.''' Ebselen.]]
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| <scene name='69/694218/Ebselen/1'>Ag85C-Ebselen</scene> (Figure 3) is characterized by a covalent bond between [http://en.wikipedia.org/wiki/Ebselen ebselen](figure 4) and Cys209, thus forcing the otherwise kinked helix α-9 to take on a relaxed conformation (Figure 3). This [http://www.jbc.org/content/289/36/25031/F4.expansion.html alteration of Cys209] allows movement of the helix and causes disruption of the hydrogen bonds within the catalytic triad, ultimately inactivating Ag85C.<ref name="Favrot"/>
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| These initial findings suggest that ebselen-like mutants characterized by alterations in Cys209 may serve as potential drug targets for ''M. tuberculosis''. Because any modification or mutation of Cys209 in Ag85C leads to either a dramatic decrease or complete loss of enzymatic activity, research suggests there is a low probability of ''M. tuberculosis'' developing resistance to a drug modifying the Cys209. The structures and results of the following mutations and modifications support a strategy for inhibiting the Ag85 complex as a whole with mechanism-based inhibitors that first react with Ser124 to promote the relaxation of helix α-9 and expose Cys209 and then react with Cys209 side chain thiol to <scene name='69/694218/Ebselen/2'>covalently modify this conserved residue</scene>. Such a bifunctional inhibitor would offer specificity while minimizing the probability of selecting for drug resistant mutants.<ref name="Favrot"/>
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| ===Ag85C-Hg===
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| [[Image:ag85c Hg zoom.png|100 xp|left|thumb|'''Figure 5.''' [http://proteopedia.org/wiki/index.php/4qdo Ag85C-Hg] active site. Glu228 and His260 are shown in red. The addition of p-chloromercuribenzoic acid disrupts the hydrogen bond between the two resides, causing a change in the helix conformation of this modified structure. The native structure is shown in green and the relaxed, modified structure is shown in blue.]]
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| [[Image:P-chloromercuribenzoic acid.jpg |100 xp|left|thumb|'''Figure 6.''' p-chloromercuribenzoic acid]]
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| The mutant [http://proteopedia.org/wiki/index.php/4qdo Ag85C-Hg] is generated with the addition of [http://en.wikipedia.org/wiki/4-Chloromercuribenzoic_acid p-chloromercuribenzoic acid] (Figure 6), the side chain of the complex is disordered due to a lack of hydrogen bonds between Glu228 and His260. Similar to what is observed in Ag85C-ebselen, the alteration in <scene name='69/694218/4qdo/1'>Ag85C-Hg</scene> relaxes the kinked helix α-9 found in the native structure of the enzyme, thus inhibiting the active site (Figure 5). The ultimate effect is a decrease to only 60% of the normal enzymatic function of Ag85C.<ref name="Favrot"/>
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| ===Ag85C-E228Q===
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| [[Image:E228Q zoom.png |100 xp|left|thumb|'''Figure 7.''' [http://proteopedia.org/wiki/index.php/4qdz Ag85C-E228Q] active site. The glutamate residue is shifted four angstroms in the mutated form of this enzyme causing a rearrangement of hydrogen bonds within the enzyme. The histidine residue, labeled in pink, takes on two conformations, binding with alternative serine residues, labeled in red.]]
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| The mutation introduced in [http://proteopedia.org/wiki/index.php/4qdz Ag85C-E228Q]causes the Glu228 of the catalytic triad to be shifted 4 angstroms from its original position in the native structure of Ag85C (Figure 7). Due to the shift of Glu228 is the loss of hydrogen bonds between Ser124 and His260. Instead, <scene name='69/694218/4qdz/2'>His260 bonds with Ser148</scene>, which also results from the shift of Glu228. A weak electron density difference in the His260 position of the native and mutated structures was also noted, suggesting that the residue may take on two alternative conformations in the <scene name='69/694218/Ag85c-e228q/1'>Ag85C-E228Q</scene> mutant. Overall, the enzyme functionality is decreased to only 17% activity.<ref name="Favrot"/>
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| Unlike other Ag85C mutants and modifications aforementioned, the <scene name='69/694218/Ag85c-e228q/1'>Ag85C-E228Q</scene>does not eliminate any hydrogen bonds in the catalytic triad. Rather, it simply replaces a carboxylate moiety with an amide. Further, the structural change observed in the low-energy conformation of the <scene name='69/694218/Ag85c-e228q/1'>Ag85C-E228Q</scene> mutant provides additional support for the hypothesis that the natively kinked helix α-9 is central to the enzymatic function of Ag85C.<ref name="Favrot"/>
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| ===Ag85C-H260Q===
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| [[Image:H260Q zoom.png|100 xp|left|thumb|'''Figure 8.''' [http://proteopedia.org/wiki/index.php/4qe3 Ag85C-H260Q] active site. A shift in helix α-9 prevents formation of any stabilizing hydrogen bonds between residues His260 and Glu228, thus decreasing its enzymatic activity.]]
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| In <scene name='69/694218/Mutationh260q/1'>Ag85C-H260Q</scene>, a shift in helix α-9 <scene name='69/694218/Ag85c-hg/1'>prevents the hydrogen bond formation</scene>between residues His260 and Glu228, thus decreasing its enzymatic activity (Figure 8). The conversion of the glutamate, a key player in the catalytic triad, to the corresponding amide-containing side chain as well as a loss of a general base in the charge relay are both key causes for the loss of function.<ref name="Favrot"/>
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| </StructureSection> | | </StructureSection> |
| == References == | | == References == |
| <references/> | | <references/> |