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| <blockquote></blockquote>{{Sandbox_Reserved_Butler_CH462_Sp2015_#}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --> | | ==Zn<sup>2+</sup> Transporter YiiP== |
| | <StructureSection load='3h90' size='340' side='right' caption='Zinc Transporter YiiP' scene=''> |
| | This is a default text for your page '''Kyle Colston/Sandbox 1'''. 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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| == ''Mycobacterium tuberculosis'' very-long-chain fatty acyl-CoA synthetase == | | ==Structure== |
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| <StructureSection load='3r44' size='340' side='right' caption='Very Long Chain Fatty Acyl CoA Synthetase (FadD13)' scene='69/694232/Opening_scene/1'> | | YiiP is a homodimer with transmembrane (TMD) and C-terminal (CTD) domains that are connected via a charge interlocking mechanism located on a flexible loop. There are 3 Zn<sup>2+</sup> binding sites per unit of homodimer. Site A is located in the TMD, site C is located in the CTD, and site B is located at the junction of the domains join. Both TMD are composed of 6 helices, 4 of which (TM1,TM2,TM4,TM5) form a pore in which Zn<sup>2+</sup> and H<sup>+</sup> can reach binding Site A. Zn<sup>2+</sup> binding at site C helps hold the CTD together and is thought to stabilize conformational changes in YiiP. |
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| = Introduction = | | ==Mechanism of Transport== |
| ''Mycobacterium tuberculosis'' very-long-chain fatty acyl-CoA synthetase, also known as <scene name='69/694233/General_pic/1'>FadD13</scene>, is unique within its class of FadD proteins in regards to its ability to house [https://en.wikipedia.org/wiki/Lipid lipid] substrates longer than itself. These lipid substrates are very-long-chain fatty acids between lengths C22 –C26, which is up to the maximum length tested. <ref name="Our Paper"/> The significance of theses very-long-chain fatty acids lies in their importance to mycolic acid synthesis by ''Mycobacterium tuberculosis'' ''(M. tb)''. Mycolic acids compose part of the cell wall of ''(M. tb)''. FadD13, an activator of mycolic acids, has been identified as key component in the virulence of [https://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''Mycobacterium tuberculosis''], the etiological agent of [https://en.wikipedia.org/wiki/Tuberculosis tuberculosis], and has emerged as possible target for novel therapeutic agents.<ref name="JT">PMID: 20454815</ref> The FadD13 enzyme is the last gene of the ''mymA'' operon. <ref name="residue paper"/>
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| There are four main groups of FadD enzymes categorized on their ability to accommodate different length substrates: short (C2-C4), medium (C4-C12), long (C12-C22), and very long (C22-C26).<ref name="Our Paper"/> Most FadD class proteins exist as integral membrane proteins, involved in both the activation of fatty acids and other hydrophobic substrates in addition to the transport of these lipids into the cell. However, substrates longer than the enzyme itself, like these very-long-chain fatty acids, pose an interesting structural dilemma to the enzyme. FadD13 differs from typical integral membrane fatty acyl-CoA synthetases in that FadD13 exists as a [https://en.wikipedia.org/wiki/Peripheral_membrane_protein peripheral membrane protein]. This key feature provides a mechanistic basis for FadD13’s activation and transport of fatty acids of length C24-C26 through the two step addition of [http://en.wikipedia.org/wiki/Coenzyme_A Coenzyme A](Figure 1).<ref name="Our Paper">PMID: 22560731</ref>
| | YiiP's ability to export Zn<sup>2+</sup> from the cytoplasm is best described as an alternating access mechanism with Zn<sup>2+</sup>/H<sup>+</sup> antiport. YiiP has 2 major structural conformations as shown by the crystallized structures [[3H90]] and [[3J1Z]] (a YiiP homolog derived from ''Shewanella oneidensis''). 3H90 shows YiiP in its outward-facing conformation and 3J1Z shows the YiiP homolog in an inward-facing conformation. |
| | When YiiP is saturated with Zn<sup>2+</sup> it seems to favor the <scene name='69/694233/Outward-facing_conformation/2'>outward-facing conformation</scene> whereas when active sites are either empty or bound to H<sup>+</sup> the <scene name='69/694233/Outward-facing_conformation/1'>inward-facing conformation</scene> is favored. This drives the export of Zn<sup>2+</sup> from the cytoplasm and enhances the coupling of the proton-motive force. Although YiiP exists as a homodimer both monomers can undergo conformation change independent of one other to produce the alternating access mechanism. |
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| = Mechanism = | | ===Zn<sup>2+</sup> Induced Conformation Change=== |
| [[Image:FadD13 edited image.jpg|425 px|left|thumb|Figure 1: Mechanism for the activation of fatty acids (C24-C26) by FadD13. The N terminal domain (pink) is embedded in the membrane with the arginine rich lid-loop (dark blue), while the flexile linker (black) connects the C terminal domain (green) to the rest of the enzyme. Activation requires the binding of ATP (blue) which induces structural changes that promote the binding of the fatty acid chain. Formation of an acyl-adenylate intermediate induces a 140° rotation of the C terminal domain and the binding of CoA (orange). ]]
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| [[Image:acyl coa synthetase.jpg|425 px|right|thumb|Figure 2: Representation of the two-step reaction catalyzed by FadD13]]
| | Conformation changes occur in the TMD and CTD, both of which are heavily influenced by the presence of Zn<sup>2+</sup>. The conformation change directly involved with Zn<sup>2+</sup>/H<sup>+</sup> antiport occurs in the TMD as helix pivoting controls what environment site A is available to. Conformation change occurs when the transmembrane helix pairs TM3-TM6 pivot around cation binding site. It is believed that the energy for TMD conformation change comes from energy of binding each substrate. Changing to the outward from the inward-facing conformation causes a shift in <scene name='69/694233/Transmembrane_helix_5/2'>TM5</scene> which disrupts the tetrahedral geometry of active site A. This in turn decreases binding affinity site A has for Zn<sup>2+</sup> making export to the periplasm possible. After Zn<sup>2+</sup> is exported and site A is either empty or bound to hydrogen change back to the inward-facing conformation is favored. |
| | In contrast the main purpose of conformation change in the CTD is to stabilize the YiiP dimer and acts as a Zn<sup>2+</sup> sensor. This is possible because of the flexible loop that links the TMD and the CTD. This loop harbors the charge interlock which serves as a hinge that allows movement of the CTD. Using FRET to measure the distance between the CTD of each monomer fluorescence quenching was observed as the concentration Zn<sup>2+</sup> increased, which supports that idea that Zn<sup>2+</sup> induces a stabilizing conformation change in the CTD. |
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| == General mechanism for the activation of fatty acids ==
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| FadD13 represents the first Fatty Acyl-CoA Synthetase of its kind to display biphasic kinetics.<ref name="residue paper"/> FadD13 first activates the fatty acid through a reaction with ATP to form an acyl adenylate intermediate and subsequently releases a pyrophosphate. Following a conformational change of the enzyme upon the binding of ATP, coenzyme A is able to bind to its active site and react with the acyl adenylate intermediate forming the [http://en.wikipedia.org/wiki/Acyl-CoA acyl CoA] product (Figure 2). These activated fatty acyl-CoA thioesters have then been demonstrated to be important for the synthesis of triacyglycerols and phospholipids in the membrane of ''Mycobacterium tuberculosis''. <ref name="residue paper"/>
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| == Structural basis for housing lipid substrates longer than the enzyme ==
| | 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. |
| The ability for FadD13 to transport and activate fatty acids of the maximum tested length C26, lies in it being a peripheral membrane protein. FadD13's attachment to the membrane via electrostatic interactions in the N-terminal domain is coupled with the presence of a hydrophobic tunnel located centrally in this same domain. This method of attachment, with the alignment of the hydrophobic tunnel to the membrane, allows the extension of these very-long-chain fatty acids to enter FadD13 from the membrane (Figure 1). Of importance to the passage of these fatty acid substrates into FadD13 resides in the presence of an arginine rich lid-loop, located at the top of the hydrophobic tunnel and embedded in the membrane. Once the lid-loop is opened, fatty acids may be pulled from the membrane into a hydrophobic tunnel, which is the main structural component by which fatty acids are capable of transportation from the membrane into the enzyme (Figure 1).
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| =Structure =
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| FadD13 is composed of 503 amino acid residues divided into three main regions: The <scene name='69/694233/N_terminal_domain/1'>N-terminal domain</scene> (residues 1-395) and <scene name='69/694233/C-terminal_domain/2'>C-terminal domain</scene> (residues 402-503) which are connected via a flexible <scene name='69/694233/Linker_section/2'>linker</scene> represented in dark blue (residues 396-401).<ref name="Our Paper"/> Each region plays an important role in the activation of fatty acids. The large N-terminal domain houses many key structural features involved in fatty acid activation, but ultimately it is the flexible linker that allows movement of the C-terminal domain to from the fully functioning active site of FadD13 (Figure 1).
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| == Electrostatics ==
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| [[Image:electrostatics fadD13.png|300 px|left|thumb|Figure 3: Pmyol depiction of electrostatic interactions of FadD13.]]
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| The electrostatics of FadD13 as seen in (Figure 3) illustrate the hydrophobic and positively charged regions that compose this protein. Experimental results revealed that the peripheral FadD13 is attached to the membrane via electrostatic and hydrophobic regions located on the top portion of the N-terminal region (Figure 3).<ref name="Our Paper"/> Of key importance in this N-terminal domain region attached to the membrane is an area of notable arginine rich residues, known as the arginine rich lid-loop.
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| == Arginine Rich Lid-loop ==
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| The <scene name='69/694233/Arginine_rich_lid_loop/1'>arginine rich lid loop</scene> functions to block entry of fatty acids into the hydrophobic tunnel of FadD13.This area on the top portion of the enzyme is also crucial in the association with the membrane as the positively charged arginine residues are attracted to the negative charge on the phospholipid heads.<ref name="Our Paper"/>
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| == Hydrophobic Tunnel ==
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| The <scene name='69/694233/Hydrophobic_tunnel/2'>hydrophobic tunnel</scene> of FadD13 is essential to the transport and accommodation of very long fatty acids from the membrane into the cell. This tunnel runs through the middle of FadD13 from the arginine rich lid loop to the ATP binding site and is situated between the and alpha helices α8-α9 and parallel beta sheet β9- β14 (Figure 4).<ref name="Our Paper"/> Negatively charged residues at the active site of FadD13 are the driving factor in the attraction of the fatty acid from the membrane through the hydrophobic tunnel of the enzyme.
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| [[Image:Hydrophobic tunnel 2.jpg|300 px|left|thumb|Figure 4: Pmyol depiction of hydrophobic tunnel.]]
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| == Active Site ==
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| The FadD13 active site is composed of positively charged regions which account for the attraction and binding of hydrophobic substrates to this region (Figure 3). The active site on FadD13 is composed of two conserved regions, one of which serves as the binding site for ATP and the other for CoA. The adenine of ATP is bound to a group of <scene name='69/694232/Adenine_binding_group/2'>six amino acids (300-305)</scene> that is structurally identically to other acyl-CoA synthetases. <ref name="Our Paper"/>
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| Mutation of the highly conserved residue in the C-terminal region, <scene name='69/694233/Lys_487/2'>Lysine 487</scene>, resulted in a 95% loss of function of FadD13 and is thought to be involved in the orientation of the substrates to form the adenylate intermediate.<ref name="residue paper">PMID: 20027301</ref> Additionally, <scene name='69/694233/Ser_404/1'>Serine 404</scene> was hypothesized to be involved in the binding of Coenzyme A which may only occur once this region incurs a 140 degree rotational change after the initial binding of ATP.<ref name="Our Paper"/><ref name="residue paper"/>
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| = Disease =
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| ''Mycobacterium tuberculosis'' ''(M.tb)'' is the causative agent involved in the disease '''tuberculosis'''. Tuberculosis is a growing global health concern that has been intensified due to the increase in HIV infections along with the increase in multi-drug resistance strains of ''(M. tb)'' <ref name="molecular studies">PMID: 20454815</ref>. Most of the drug resistance has evolved due to the intensive nature of the treatment for tuberculosis, which often goes incomplete thus resulting in drug resistant strains; therefore, the importance in identifying characteristics and residues to be exploited for new drug targets is pivotal <ref name="molecular studies"/>.
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| The cell wall of ''(M. tb)'' is known to be composed and synthesized from a distinct variety of lipids, most notably [https://en.wikipedia.org/wiki/Mycolic_acid mycolic acids], which are known to play a crucial role in the pathogenesis of ''(M. tb)'' <ref name="molecular studies"/> The mycolic acid biosynthetic pathway has been proposed to involve five distinct stages, the first of which is the synthesis of C20 to C26 straight-chain saturated fatty acids activated by FadD13. <ref name="Drug Inhibitors">PMID: 12164478</ref> For this reason, current research has focused on inhibitors to disrupt the initiation of this biosynthetic pathway of mycolic acids in ''(M. tb)''.
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| == Current Treatment ==
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| Drugs developed thus far that have been shown to inhibit mycolic acid biosynthesis are: [https://en.wikipedia.org/wiki/Isoniazid isoniazid], [https://en.wikipedia.org/wiki/Ethionamide ethionamide], [https://en.wikipedia.org/wiki/Thiocarlide thiocarlide], thiolactomycin, and [https://en.wikipedia.org/wiki/Triclosan triclosan]. <ref name="Drug Inhibitors"/> Additionally, [https://en.wikipedia.org/wiki/Pyrazinamide pyrazinamide] was shown to inhibit fatty acid synthase type I which is involved in providing a precursor necessary for fatty acid elongation to long-chain mycolic acids. <ref name="Drug Inhibitors"/> Treatment for active cases of tuberculosis include the simultaneous therapeutic use of two or more frontline drugs: isoniazid, ethambutol, rifampicin and pyrazinamide. <ref name="molecular studies"/>
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| == Future Research ==
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| While currently there are no specific drug targets for FadD13, a better understanding of key residues involved in the activation of very-long-chain fatty acids is a promising start to developing new drug targets for ''(M. tb)''. Recent studies have shown the ''mymA'' operon, which is involved in the maintenance of ''(M. tb)'' cell wall architecture, and which codes for the enzyme FadD13, is up-regulated under acidic conditions. <ref name="molecular studies"/> Functional loss of the ''mymA'' operon resulted in increased drug sensitivity and death of the pathogen; therefore any drugs that can can target this operon will be effective at fighting tuberculosis. <ref name="molecular studies"/>
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| </StructureSection> | | </StructureSection> |
| | | == References == |
| __NOTOC__
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| ==References== | |
| <references/> | | <references/> |
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| ==Similar Proteopedia Pages==
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| [http://proteopedia.org/wiki/index.php/Fatty_acid_synthase Fatty Acid Synthase]
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| [http://www.proteopedia.org/wiki/index.php/Molecular_Playground/4%27-PHOSPHOPANTETHEINYL_TRANSFERASE_%28Sfp%29 Phosphopantetheinyl Transferase]
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| [http://proteopedia.org/wiki/index.php/Acyl_carrier_protein Acyl Carrier Protein]
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| [http://www.proteopedia.org/wiki/index.php/Lipase Lipase]
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| ==External Resources==
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| [https://en.wikipedia.org/wiki/Lipid Lipids] Wikipedia page
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| [https://en.wikipedia.org/wiki/Tuberculosis Tuberculosis] Wikipedia page
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| [https://en.wikipedia.org/wiki/Mycobacterium_tuberculosis ''Mycobacterium tuberculosis''] Wikipedia page
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| [http://en.wikipedia.org/wiki/Coenzyme_A Coenzyme A] Wikipedia page
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| [http://en.wikipedia.org/wiki/Acyl-CoA Acyl CoA] Wikipedia Page
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| [https://en.wikipedia.org/wiki/Mycolic_acid Mycolic Acid] Wikipedia page
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| [https://en.wikipedia.org/wiki/peripheral_membrane_protein Peripheral Membrane Protein] Wikipedia page
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| [https://en.wikipedia.org/wiki/Ethionamide Ethionamide] Wikipedia page
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| [https://en.wikipedia.org/wiki/Isoniazid Isoniazid] Wikipedia page
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| [https://en.wikipedia.org/wiki/Thiocarlide Thiocarlide] Wikipedia page
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| [https://en.wikipedia.org/wiki/Triclosan Triclosan]
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| [https://en.wikipedia.org/wiki/Pyrazinamide Pyrazinamide] Wikipedia page
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