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| - | =='''Caspase 3- 1rhk'''== | + | =='''Estrogen receptor beta/p-hydroxybenzene sulfonamide complexes (2yly)<ref>PMID: 21885279 </ref>'''== |
| | + | by Benjamin Homyak, Soo Lim Park, Marissa Burgess |
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| - | ===Introduction=== | + | [[Student Projects for UMass Chemistry 423 Spring 2016]] |
| | + | <StructureSection load='2yly' size='350' side='right' caption='p-hydroxybenzenesulphonamides ERb receptor (PDB entry [[2yly]])' scene=''> |
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| - | Caspases are a group of dimeric cysteine proteases that play important roles to control the ultimate steps of apoptosis and innate inflammation; they are also very important in cellular development, homeostasis and in a wide range of diseases such as neurodegeneration, ischemia and cancers. During apoptosis, the initiator caspases (caspases 8 and 9) as upstream regulators cleave and activate with the downstream executioner caspases (caspases 3, 6 and 7). Then the activated executioner caspases will cleave upwards of 500 key proteins and DNA, which finally cause the death of cells.<ref>Jeanne A. Hardy and James A. Wells, Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes, ''THE JOURNAL OF BIOLOGICAL CHEMISTRY'',VOL. 284, NO. 38, pp. 26063–26069, September 18, 2009.http://www.jbc.org/content/284/38/26063.short</ref>
| + | ==Introduction== |
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| - | [[Image:Caspase mediated apoptosis.PNG]]<Structure load='1rhk' size='500' frame='true' align='right' caption='Two heterodimers(one in light green, one in light blue) of Caspase 3 interact together to form the full 12-stranded beta-sheet (inner arrows) structure surrounded by alpha-helices (outer helices) that is unique to caspases.' scene='Insert optional scene name here'/>
| + | Estrogen receptors are proteins found on and inside of the cell. When activated by estrogen these receptors are important in sexual maturation and gestation. There are two types of estrogen receptors that exist which include nuclear estrogen receptors (ERα and ERβ), which are included in the nuclear receptor family of intracellular receptors, and membrane estrogen receptors. |
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| - | There are many caspase structures are researched, and most of them involve either peptide, protein inhibitors or unattractive candidates for drug development.<ref>Crystal structure of the complex of caspase-3 with a phenyl-propyl-ketone inhibitorReducing the peptidyl features of caspase-3 inhibitors: a structural analysis.Becker, J.W., Rotonda, J., Soisson, S.M., Aspiotis, R., Bayly, C., Francoeur, S., Gallant, M., Garcia-Calvo, M., Giroux, A., Grimm, E., Han, Y., McKay, D., Nicholson, D.W., Peterson, E., Renaud, J., Roy, S., Thornberry, N., Zamboni, R.,Journal: (2004) J.Med.Chem. 47: 2466-2474</ref> <scene name='Sandbox_Reserved_433/Initial_view/3'>Caspase 3</scene> is one of the downstream executioner caspases which interacts with caspase 8 and 9. It is formed from a 32 kDa zymogen that is cleaved into 17 kDa and 12 kDa subunits. It contains four anti-parallel beta sheets from p17 and two from p12, which will come together to make a heterodimer that will interact with another heterodimer to form a total 12-stranded <scene name='Sandbox_Reserved_433/Beta_sheets/1'>beta sheets</scene> structure surrounded by <scene name='Sandbox_Reserved_433/Beta_sheets/2'>alpha-helices</scene> that is owned by caspases only.<ref>Salvesen GS (January 2002). "Caspases: opening the boxes and interpreting the arrows". Cell Death Differ. 9 (1): 3–5. doi:10.1038/sj.cdd.4400963. PMID 11803369.</ref><ref>Lavrik IN, Golks A, Krammer PH (October 2005). "Caspases: pharmacological manipulation of cell death". J. Clin. Invest. 115 (10): 2665–72. doi:10.1172/JCI26252. PMC 1236692. PMID 16200200</ref> As an executioner caspase, the caspase-3 zymogen does not have activity until it is cleaved by an initiator caspase after apoptotic signaling events have occurred.<ref>Walters J, Pop C, Scott FL, et al. (December 2009). "A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis". Biochem. J. 424 (3): 335–45. doi:10.1042/BJ20090825. PMC 2805924. PMID 19788411</ref> Caspase 3 has many of the typical characteristics which all currently-known caspases also own. For instance, its <scene name='Sandbox_Reserved_433/Active_sites/4'>active site</scene> contains a cysteine residue, <scene name='Sandbox_Reserved_433/Cys_285/4'>Cys-285</scene> and histidine residue, <scene name='Sandbox_Reserved_433/His_237/3'>His-237</scene>. Also, Caspase 3 is able to be activated by diverse death-inducing signals, which includes the chemotherapeutic agents. Pathways of the activation of Caspase 3 was already identified that were either dependent on or independent of mitochondrial cytochrome c release and Caspase 9 function. It also plays an significant role many other apoptotic scenarios in tissue-, cell type-, or death stimulus-specific manner.<ref>Emerging roles of caspase-3 in apoptosis,Alan G Portera and Reiner U Jänicke,Institute of Molecular and Cell Biology, The National University of Singapore, 30 Medical Drive, Singapore 117609, Republic of Singapore: http://www.nature.com/cdd/journal/v6/n2/abs/4400476a.html</ref>
| + | A series of p-hydroxybenzenesulphonamides ERβ receptor agonists discovered along with various compounds listed showed selectivity over the ERα receptor. Overall, they found that compound 11 showed better binding conformation determined by X-ray, and presents a better starting point for the journey to find a more selective ERβ agonist. |
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| | + | Looking at the gonadal steroid hormone estradiol,1, action is performed through estrogen subtypes, ERα and ERβ. The detrimental effects of ERβ in comparison to the proliferative effects ERα are found to inhibit breast and endometrial tissue compared to ERα and could potentially be responsible for the immunomodulatory and neuropharmacalogical behavior of estradiol 1,2. The interest in the therapeutic benefits of selective ERβ agonists to combatant various conditions including endometriosis and inflammatory bowel disease. |
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| - | Since Caspase 3 cleaves a wide range of cellular substrates including structural proteins and DNA repair enzymes and also activates an endonuclease caspase-activated DNAse, which causes the DNA fragmentation that is characteristic of apoptosis, it has many biological functions, such as normal brain development and several significant diseases including Alzheimer’s disease, Polycystic Kidney Disease and Cancers. In a very frequently-occurring cancer, breast cancer, Caspase 3 acts as a key mediator whose mRNA and protein expression was examined, which leads that the rates of apoptosis as measured by both caspase 3 activation and nucleosome release are higher in breast cancer than in nonmalignant breast tissue.<ref>Caspase 3 in breast cancer.,O'Donovan N, Crown J, Stunell H, Hill AD, McDermott E, O'Higgins N, Duffy MJ.
| + | This is an overall scene with the beta sheets in purple and the alpha helices in ball and stick figures <scene name='48/483890/Ben_homyak_overall_structure/1'>Overall Structure</scene> |
| - | Source, Department of Medical Oncology, St. Vincent's University Hospital, University College Dublin, Dublin 4, Ireland. http://www.ncbi.nlm.nih.gov/pubmed/12576443</ref> In another cancer, lung cancer, researches shown that polymorphisms in the caspase 3 gene may influence caspse 3 production and activity, thereby modulating the susceptibility to lung cancer.<ref>Identification of Polymorphisms in the Caspase-3 Gene and Their Association With Lung Cancer Risk
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| - | Jin Sung Jang,Kyung Mee Kim,Jin Eun Choi,Sung Ick Cha,Chang Ho Kim,Won Kee Lee,Sin Kam,Tae Hoon Jung and Jae Yong Park, Department of Biochemistry, School of Medicine, Kyungpook National University, Daegu, Korea. http://onlinelibrary.wiley.com/doi/10.1002/mc.20397/pdf</ref> Thus, to cure cancers, studying on Caspase 3 becomes a significant entrance.
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| | + | Here is another scene with a rainbow diagram description of the whole molecule |
| | + | <scene name='48/483890/2yly_overall_diagram/3'>Rainbow diagram</scene> |
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| | + | ==Overall Structure== |
| | + | The assembly composition of the 2yly protein is a homodimer. (Quaternary structure) |
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| | + | The DNA Binding domain is located in the C region, which is highly conserved. The ligand binding domain is located at the C terminus in the E and F regions. The ligand binding domain is often called the “three-layered anti-parallel α helical sandwich” because it contains 12 alpha helices along with one beta hairpin. Sites for dimerization and nuclear localization are located in the the D region, which is poorly conserved. Both ERα and ERβ have several splice variants, with ERα having over 20 and ERβ having 5. |
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| | + | The 2yly protein's secondary structure consists of mostly alpha helices (148 residues) and only two beta strands (6 residues) on the outside of the receptor shown below with the alpha helices showing polar and non-polar parts of the chain. The beta sheets are shown in yellow in the second green scene. |
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| - | ===Overall Structure=== | + | - <scene name='48/483890/2yly_alpha_helicies/1'>Alpha Helices</scene>, and <scene name='48/483890/2yly_beta_sheets/1'>Beta Sheets</scene> |
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| - | <Structure load='1RHK' size='500' frame='true' align='right' caption='Caspase-3' scene='Insert optional scene name here' />
| + | - Tertiary Structure |
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| | + | - Polar- Pink, Hydrophobic- Grey <scene name='48/483890/2yly_polar_hydrophobic/1'>Polar and Nonpolar Groups</scene> |
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| - | Caspase 3, as shown here, is composed of three chains (referred to as <span style="color:blue">'''A (p17)'''</span>, <span style="color:green">'''B(p12)'''</span>, and <span style="color:pink">'''C'''</span>) that are 147, 102, and 5 amino acids long (respectively). The three chains are primarily held together through a parallel non-polar beta-strand connection between chains A and B shown <scene name='Sandbox_Reserved_433/A-b/1'>here</scene>, and an anti-parallel polar beta-strand connection between chains B and C shown <scene name='Sandbox_Reserved_433/B-c/1'>here</scene>. Chain A consists of 7 beta-strands and 4 alpha helixes. Chain B has 4 beta-strands and 3 alpha-helices. 6 beta-strands (4 from chain A, and 2 from chain B) form a beta-sheet in the middle of the enzyme surrounded by alpha-helices. This <scene name='Sandbox_Reserved_433/Secondary/1'>structure</scene> (<span style="color:purple">'''alpha-helix'''</span>, <span style="color:gold">'''beta-strand'''</span>) causes defining characteristic of caspases and is caused by hydrophobic interactions due to the polarity of the structures shown <scene name='Sandbox_Reserved_433/Polarity/9'>here</scene> (<span style="color:grey">'''hydrophobic'''</span>, <span style="color:purple">'''polar'''</span>).
| + | - Surface groups (orange) vs. Buried groups (blue) <scene name='48/483890/2yly_surface_buried/1'>Check it out</scene> |
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| | + | ==Binding Interactions== |
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| - | Only chains A and B are conserved. Chain C consists of a single beta-sheet which can be of variable length and sequence which, though close to the active site on chain A does not seem to be part of it. This is why, though there are three chains shown here, Caspase-3 is considered a heterodimer, and not a heterotrimer. The following example of Caspase-3 with a bromomethoxyphenyl inhibitor does not have a chain C, the inhibitor is considered part of chain A.
| + | Among the series of p-hydroxybenzene sulfonamide ERβ receptor agonists discovered, protein 2yly has been identified for great selectivity over the related ERα receptor. Protein 2yly was originally designed to form an interaction with the His 475 through the tertiary hydroxyl group. However, the hydroxyl group serves as a conformational lock to form an internal hydrogen bond with the <scene name='48/483890/Soolim_binding/1'>sulphonamide oxygen </scene> about 2.3 Å apart.<sup>[1]</sup> |
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| | + | Internally packed against the phenyl group is the cyclopropyl group with large <scene name='48/483890/Soolim_hydrophobic/1'>hydrophobic interactions</scene> (shown in gray) between Ile 373 and both the benzyl and chiral methyl groups. The sulphonamide oxygens come in close contact with Met 336 and the benzyl group of the ligand comes near His 475 but there are no coulombic interactions formed. Depending on the functional group placed either next to the tertiary hydroxyl group or on the benzyl ring, further Van der Waals interactions can be seen within the lipophilic pocket. In other words, depending on the polarity of the group attached to the sulphonamide, interactions with His 475 will be varied. |
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| - | The structure shown here is <scene name='Sandbox_Reserved_433/1rhq_2/1'>CASPASE-3 WITH A BROMOMETHOXYPHENYL INHIBITOR</scene> | |
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| | + | ==See Also== |
| | + | *[[http://www.proteopedia.org/wiki/index.php/Estrogen_receptor Estrogen Receptor]] |
| | + | *[[http://www.proteopedia.org/wiki/index.php/1yy4 1yy4]] |
| | + | *[[http://www.proteopedia.org/wiki/index.php/1u3s 1u3s]] |
| | + | *[[http://www.proteopedia.org/wiki/index.php/1x78 1x78]] |
| | + | *[[http://www.proteopedia.org/wiki/index.php/1qkm 1qkn]] |
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| | + | ==Credits== |
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| - | While the heterodimer shown above and below is the active complex; the original <scene name='Sandbox_Reserved_433/1rhq/4'>heterodimer</scene> of casepase-3 will form a heterotetramer through an anti-parallel beta sheet interaction with another caspase-3 through each final beta-strand in chain B shown <scene name='Sandbox_Reserved_433/Chain_b_connection/1'>here</scene>. This forms a <scene name='Sandbox_Reserved_433/Secondary_structure/1'>heterotetramer</scene> containing a non-polar twelve stranded beta sheet surrounded by polar non-polar alpha-helixes is a defining characteristic of caspases, shown <scene name='Sandbox_Reserved_433/2-secondary/1'>here</scene> (polarity shown <scene name='Sandbox_Reserved_433/Polarity-2/1'>here</scene>, <span style="color:grey">'''hydrophobic'''</span>, <span style="color:purple">'''polar'''</span>) , particularly caspases-1, 3, 7, and 8.<ref>Salvesen GS "Caspases: opening the boxes and interpreting the arrows" Nature Publishing Group 2002 vol:9 iss:1 pg:3 -5</ref> thank you!
| + | Introduction - Benjamin Homyak |
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| - | ===Binding Interactions===
| + | Overall Structure - Marissa Burgess |
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| - | <Structure load='1rhk' size='500' frame='true' align='right' caption='1rhk of Caspase 3 has a variety of inhibitors with which it interacts with take advantage of binding to the active site which prevents the substrate from binding.' scene='Insert optional scene name here' />
| + | Drug Binding Site - Soo Lim Park |
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| - | The <scene name='Sandbox_Reserved_433/Acitve_site_caspase3/1'>catalytic site</scene> of the enzyme has been determined to be Cys-285 and His-237. Cys-285 can be seen with its sulfur group and yellow and the unique nitrogen-carbon ring of His-237 is seen in gray and blue. His-237 stablizes the peptide while Cys-285 attacks, cleaving the peptide bond of peptides with sequences Asp-X-X-Asp. In addition to the active site, Cys-285 and Gly 238 stabilize the transition state through hydrogen bonding and therefore also very important. There is a
| + | ==References== |
| - | <scene name='Sandbox_Reserved_433/Acitve_site_caspase3/3'>specificity pocket</scene> to stabilize the Asp residue of the peptide undergoing cleavage. The pocket,which can be seen as the pink amino acids, consists of polar amino acids such as Arg, Ser, and Gln which allows polar Asp to be held in the correct orientation for the reaction to occur <ref>Cohen G. M. (1997). Caspases: the executioners of apoptosis. Biochem. J. 326(Pt 1), 1–16.short</ref> .
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| - | Since the catalytic site of the molecule has been determined, inhibitors have been developed which have the ability to bind to these essential amino acids to prevent the enzyme from working. Caspase 3 is regulated with small peptides and other proteins in vivo but since proteins are often large and need specific conditions to work, they are often not ideal to use for medical treatments.
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| - | There are numerous inhibitors available commericially as well as research for more specific and easily controlled inhibitors. Ac-DEVD-aldehyde is a commercially available inhibitor of Caspase 3.
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| - | <scene name='Sandbox_Reserved_433/Binding_site_inhibitor_1/3'>Ac-DEVD-aldehyde binding</scene> interacts directly with the Cys 285 of the active site leading to the formation of a hemithioacetal and stablization by hydrogen bonding with Cys 285 and Gly 238 <ref>Becker JW, Rotonda J, Soisson SM, Aspiotis R, Bayly C, Francoeur S, Gallant M, Garcia-Calvo M, Giroux A, Grimm E, Han Y, McKay D, Nicholson DW, Peterson E, Renaud J, Roy S, Thornberry N, Zamboni R. Reducing the peptidyl features of caspase-3 inhibitors: a structural analysis. J Med Chem. 2004;47:2466–2474.short</ref>. This inhibits Caspase 3 through competetive inhibition since it binds to the same site as the substrate. All of the colored amino acids in the green scene show the binding sites of the inhibitor with the pink amino acids signifiying the catalytic diyad. This convention will stay throughout all of the inhibitor binding scenes.
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| - | Other molecules such as nicotinic acid aldehydes have also been seen to inhibit Caspase 3 and have shown great promise due to peptide-less characteristics. In experimental trials nicotinic acid aldhyde A had a similar
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| - | <scene name='Sandbox_Reserved_433/Inhibitor7/1'>binding region</scene> as Ac-DEVD-aldehyde binding directly to the catalytic diad and preventing substrate binding. However there are other nicotinic acid aldhydes that have <scene name='Sandbox_Reserved_433/Inhibitor8/1'>additional binding regions</scene> and have shown to have greater inhibition. For example both nicotinic acid aldegydes have strong interactions with Tyr 388 and Phe381 which lead researchers to believe that these amino acids also have an important role in the catalytic mechanism of the enzyme. This reinforces the idea that there are many parts of the protein that are vital for correct function and shape, not exclusively the active sites <ref>Becker JW, Rotonda J, Soisson SM, Aspiotis R, Bayly C, Francoeur S, Gallant M, Garcia-Calvo M, Giroux A, Grimm E, Han Y, McKay D, Nicholson DW, Peterson E, Renaud J, Roy S, Thornberry N, Zamboni R. Reducing the peptidyl features of caspase-3 inhibitors: a structural analysis. J Med Chem. 2004;47:2466–2474.short</ref>.
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| - | After creating a list of <scene name='Sandbox_Reserved_433/Common_sites/2'>common binding sites</scene> for multiple inhibitors, it can been seen that the most important region of binding is around the catalytic diyad. Each of the common amino acids involved in binding it highlighted in a different color and illustrates the importance of the availability of this region for proper function.
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| - | ===Additional Features===
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| - | <Structure load='1f1j' size='500' frame='true' align='right' caption='The activation of procaspase-7 brings a cysteine residue onto the surface' scene='Insert optional scene name here' />
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| - | '''Activation'''
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| - | Since the function of caspase-3 is so crucial to the death of the cell, having it active all of the time would be highly detrimental to the survival of the cell. As mentioned before, the cell gets around this issue by expressing caspase-3 as an inactive proenzyme called procaspase-3. In the apoptotic pathways, procaspase-3 is activated both by caspase-9 as well as caspase-3 <ref>Fan, Ting-Jun, Han, Li-Hui, Cong, Ri-Shan, and Liang, Jin. “Caspase Family Proteases and Apoptosis”. Acta Biochimica et Biophysica Sinica. Vol. 37 No. 11 (2005): 719 – 27. Web. http://www.abbs.info/pdf/37-11/1-719-727-05165.pdf </ref>. This is a good example of enzyme regulation; since having more caspase-3 is good for apoptosis activation of procaspase-3 by caspase-3 itself is something very useful for the cell to do.
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| - | The most important thing for procaspase-3 to do, however, is block itself from being casually and unintentionally activated. This is accomplished through use of an internal switch of sorts present on the proenzyme that acts as a safety, not unlike a gun. This switch on procaspase-3 is comprised of three Aspartic Acid residues near the intersection of the p17 and p12 subunits <ref>Porter, Alan G. “Flipping the safety catch of procaspase-3”. Nature Chemical Biology. Vol. 2. No. 10 (2006): 509 – 10. Web. http://www.nature.com/nchembio/journal/v2/n10/full/nchembio1006-509.html
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| - | </ref>. These three residues, Asp 179 – Asp 181, are located near the active site of the enzyme, Cys 163. By removing or modifying this DDD safety, either by an upstream signal or by targeted drugs, procaspase-3 can be converted into caspase-3, thereby activating it.
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| - | Unfortunately, the structure for procaspase-3 is not on Proteopedia at all. Luckily for us, caspase-7 undergoes a similar sort of activation (and the structure of both caspase-7 and its proenzyme) are both available. As with caspase-3, the active site of caspase-7 is a cysteine residue, here Cys 186, located on the outside of the molecule. Seen in orange <scene name='Sandbox_Reserved_433/Active_caspase-7/2'>here</scene>, the presence of this cysteine catalyzes the cleavage reaction that caspase proteins are known for. The residues in black, Arg 187 and Tyr 223, are allosterically stabilized, and the residue in light blue, Gly 188, acts as a hinge of sorts to get the two allosterically stabilized residues in place <ref>
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| - | Hardy, Jeanne A. and Wells, James A. “Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes” The Journal of Biological Chemistry. Vol. 284. No. 38 (2009): 26063 – 69. Web. http://www.jbc.org/content/284/38/26063.full</ref>. If we look at the
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| - | <scene name='Sandbox_Reserved_433/Procaspase_7_active_site/2'>proenzyme form</scene> of caspase-7, we see that the allosterically linked residues are actually rather far apart. This keeps Cys 186 away from the surface of the enzyme and keeps it inactive.
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| - | While the activation in procaspase-3 is slightly different, as it involves the removal of as safety catch of sorts, the key idea to take home is that in order for caspases to function, they must be activated. The activation of these caspases leads directly of the exposure of the cysteine that makes up the active site.
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| - | ===Credits===
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| - | Introduction - Di Lin
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| - | Overall Structure - Austin Virtue
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| - | Drug Binding Site - Jill Moore
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| - | Additional Features - Alex Way
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| - | ===References===
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| | <references/> | | <references/> |
