Sandbox Reserved 763: Difference between revisions

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The crystal structure of [[2iyq]] from the Protein Data Bank <ref name="PDB">Protein Data Bank http://www.rcsb.org/pdb/explore.do?structureId=2IYQ</ref> showing shikimate kinase from ''Mycobacterium tuberculosis'' complexed with ADP and shikimate is shown to the right as the <scene name='56/564039/Defaultscene/1'>default scene</scene>. In addition, the 2D model of [[2gij]], showing the space filling details of the asymmetric unit of MtSK, is shown to the left.  
The crystal structure of [[2iyq]] from the Protein Data Bank <ref name="PDB">Protein Data Bank http://www.rcsb.org/pdb/explore.do?structureId=2IYQ</ref> showing shikimate kinase from ''Mycobacterium tuberculosis'' complexed with ADP and shikimate is shown to the right as the <scene name='56/564039/Defaultscene/1'>default scene</scene>. In addition, the 2D model of [[2gij]], showing the space filling details of the asymmetric unit of MtSK, is shown to the left.  
===3D Structures in Different Organisms===
===3D Structures in Different Organisms===
Crystal structure have been reported for SK alone, as  a binary complex, and as a ternary complex.
''Mycobacterium tuberculosis''
''Mycobacterium tuberculosis''


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[[1iyw]] - MtSK (open lid) + ATP
[[1iyw]] - MtSK (open lid) + ATP


[[2g1k]], [[2iyr]],  [[2iyx]] - MtSK + shikimate
[[2g1k]], [[2iyr]] - MtSK + shikimate


[[2iys]] - MtSK (open lid) + shikimate
[[2iys]] - MtSK (open lid) + shikimate
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[[2dfn]], [[1we2]], [[2iyq]], [[1u8a]] - MtSK + ADP + shikimate
[[2dfn]], [[1we2]], [[2iyq]], [[1u8a]] - MtSK + ADP + shikimate
[[2iyx]] - MtSK + shikimate + sulfate


[[2iyz]] - MtSK + ADP + shikimate-3-phosphate
[[2iyz]] - MtSK + ADP + shikimate-3-phosphate
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[[1zyu]] - MtSK + AMPPCP + shikimate  
[[1zyu]] - MtSK + AMPPCP + shikimate  




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Through experiments involving size exclusion liquid chromatography and gel filtration chromatography, the oligomeric state of homogeneous MtSK was found. The molecular mass was found to be 20.7 kDa and when compared to the suggested value of 18.5 kDa, it was found that MtSK is a monomer in solution.<ref>The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918</ref>
Through experiments involving size exclusion liquid chromatography and gel filtration chromatography, the oligomeric state of homogeneous MtSK was found. The molecular mass was found to be 20.7 kDa and when compared to the suggested value of 18.5 kDa, it was found that MtSK is a monomer in solution.<ref>The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061918</ref>


===Active Residues===
===Domains and Active Residues===
[[Image:ActiveResidues2iyq.PNG|300px|left|thumb| Binding Sites<ref name="PDB" />]]
There are three domains present in Shikimate kinase: the CORE domain, and substrate-binding (SB) domain, and LID domain. Each domain plays a role in binding and is related to the active residues. The binding site for nucleotides is in the CORE, shikimate binds in the substrate-binding domain, and once ATP or shikimate binds, the LID domain closes over the active site.<ref name="UniProt">UniProt http://www.uniprot.org/uniprot/P0A4Z2</ref>
Using [[2iyq]] as an example, the active residues were binding in shikimate kinase occurs is shown to the left. There are three domains present in Shikimate kinase: the CORE domain, and substrate-binding (SB) domain, and LID domain. Each domain plays a role in binding and is related to the active residues. The binding site for nucleotides is in the CORE, shikimate binds in the substrate-binding domain, and once ATP or shikimate binds, the LID domain closes over the active site.<ref name="UniProt">UniProt http://www.uniprot.org/uniprot/P0A4Z2</ref>


The CORE domain spans from residues 9-17 (phosphate binding loop), 148-155 (AB-loop), and 101-110 (segment which includes alpha6 from 104-110). The SB domain is comprised of residues 32-93 and consists of a sub-domain from residues 32-61 which relates to the NMP-binding domain in NMP kinases. Finally, the LID domain consists of residues 112-124. A global motion leads to LID flapping over the active site upon binding the first substrate (whether shikimate or nucleotide). This causes a change from the open to closed conformation. <ref name="miscinfo">Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283606011685#</ref>
The CORE domain spans from residues 9-17 (phosphate binding loop), 148-155 (AB-loop), and 101-110 (segment which includes alpha6 from 104-110). The SB domain is comprised of residues 32-93 and consists of a sub-domain from residues 32-61 which relates to the NMP-binding domain in NMP kinases. Finally, the LID domain consists of residues 112-124. A global motion leads to LID flapping over the active site upon binding the first substrate (whether shikimate or nucleotide). This causes a change from the open to closed conformation. <ref name="miscinfo">Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0022283606011685#</ref>
Understanding the active residues involved in ligand binding is essential to design inhibitors. The P-loop of SK is often targeted by inhibitors as the ATP-binding site catalyzes the reaction (the transfer of a phosphate group). SK binds shikimate at R57, R116, and R132. The hydroxyl groups present on shikimate form contact with M10, D33, G79-81, E114, V44, F48, and R116.  The most critical residues for SK catalysis include D33, F48, R57, R116, and R132. It is believed that R57, R132, and F48 form a favorable environment to interact with shikimate, thus triggering a series of  conformational changes. Catalyzing the phosphorylation of the 3-hydroxyl group of shikimate is initiated as R116 contacts shikimate during a conformational change.<ref name="compinhibitor"> Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism. PLoS One.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306394/#!po=19.5652</ref>




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SO4
<scene name='56/564039/Sulfate/1'>SO4</scene> Sulfate is replaced with MgATP in the ternary complex of SK to allow for the reaction to occur.<ref name="sulfate"> Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis. Journal of Molecular Biology. http://www.sciencedirect.com/science/article/pii/S0022283606011685 </ref> SO4 interacts with Arg117 and distorts the nucleotide binding site<ref name="miscinfo" />


<scene name='56/564039/Mgadpligand/1'>MgADP</scene>
<scene name='56/564039/Mgadpligand/1'>MgADP</scene>
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SO4 interacts with Arg117 and distorts the nucleotide binding site<ref name="miscinfo" />
 


ADP interacts with P-loop, AB-loop, and alpha6<ref name="miscinfo" />
ADP interacts with P-loop, AB-loop, and alpha6<ref name="miscinfo" />
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Ligands associated with shikimate kinase include ADP, CL, shikimate (SKM), and TRS.
Ligands associated with shikimate kinase include ADP, CL, shikimate (SKM),


Adenosine-5'-disphosphate (<scene name='56/564039/Adpligand/1'>ADP</scene>
Adenosine-5'-disphosphate (<scene name='56/564039/Adpligand/1'>ADP</scene>
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===Methods Used to Solve===
===Methods Used to Solve/Study===
The following methods used in solving for the structure of SK include but are not limited to:
Methods used in solving and/or studying the structure of SK include but are not limited to:


Crystallization via hanging-drop vapor-diffusion
Crystallization (hanging-drop vapor-diffusion)


Molecular replacement methods for structure refinement  
Molecular replacement methods for structure refinement  


multiple isomorphous replacement
Multiple isomorphous replacement


Fourier calculations
Fourier calculations
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Chromatography
Chromatography
Site-directed mutagenesis
Isothermal titration calorimetry




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<ref name="miscinfo" /><ref name="inhibitors" />
<ref name="miscinfo" /><ref name="inhibitors" />


Studies of potential inhibitors of SK have been conducted. This is important in the identification of potential drugs against tuberculosis (TB), a curable infectious disease found mainly in developing countries. TB is curable, yet one hundred million people are infected annually with approximately three million resulting deaths. TB resurges as a health problem due to multi-drug resistant, extensively drug resistant, and totally-drug resistant strains of ''Mt''.<ref name="inhibitors" />
Studies of potential inhibitors of SK have been conducted. This is important in the identification of potential drugs against tuberculosis (TB), which is caused by the pathogen ''Mycobacterium tuberculosis.'' TB is a curable infectious disease found mainly in developing countries. Though TB is curable, 100 million people are infected annually with approximately three million resulting deaths. TB resurges as a health problem due to multi-drug resistant, extensively drug resistant, and totally-drug resistant strains of ''Mt''.<ref name="inhibitors" />Disrupting the gene encoding MtSK, ''aroK'', suggests that the shikimate pathway holds great promise for antimicrobial drug discovery.<ref name="compinhibitor" />
 
 
One way potential MtSK inhibitors have been studied is through molecular docking simulations experiments. These experiments predict the conformation of a receptor-ligand complex to analyze all possible positions of the ligand to  make a selection for the best position. Virtual screening was also used to identify active molecules compared to a specific protein target. These studies have been able to confirm staurosporine as a known SK inhibitor.<ref name="inhibitors" />
 
===Antimicrobial Agents===
===Herbicides===
===Inhibitors===
===Anti-parasitic Drug Target===
===Medical Importance===
Medical importance - related disease, if used as drug target.


One way potential MtSK inhibitors have been studied is through molecular docking experiments. These experiments predict the conformation of a receptor-ligand complex to analyze all possible positions of the ligand to  make a selection for the best position. Virtual screening was also used to identify active molecules compared to a specific protein target. These studies have been able to confirm staurosporine as a known SK inhibitor.<ref name="inhibitors" />
Through docking studies, it has been found that <scene name='56/564039/Nsc162535/1'>NSC162535</scene> is a competitive inhibitor for SK.<ref name="compinhibitor" /> This inhibitor causes a shift in the SB domain due to E53 and R132 interacting instead of E53 and R57 as seen in the wild-type. The geometry of the active site is altered, leading to inhibition of SK.<ref name="compinhibitor" />


Other uses (ie antibiotics etc)


==References==
==References==
{{reflist}}
{{reflist}}
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