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=='''Physical Models of Acetylcholinesterase in Complex with Acetylcholine and the Green Mamba Snake Toxin, Fasciculin-II'''==
=='''Acetylcholinesterase:  A Story of Substrate Traffic and Inhibition'''==
Students: Mary Acheampong. Daviana Dueño, Bobby Glover, Alafia Henry, Randol Mata, and Marisa VanBrakle, Hostos-Lincoln Academy.
Students: Mary Acheampong. Daviana Dueño, Bobby Glover, Alafia Henry, Randol Mata, and Marisa VanBrakle, Hostos-Lincoln Academy.


Teacher: Allison Granberry, Hostos-Lincoln Academy
Teacher: Allison Granberry, Hostos-Lincoln Academy


Mentors: Joel Sussman, Weissman Institute of Science, and Lars Westblade, Touro College of Pharmacy.
Mentors: Joel L. Sussman, Weizmann Institute of Science, and Lars Westblade, Touro College of Pharmacy.
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==='''Introduction'''===
==='''Introduction'''===


Acetylcholinesterase(AChE) is essential for the hydrolysis of the neurotransmitter acetylcholine(ACh), and therefore the termination of the nerve impulse in cholinergic synapses. Irreversible inhibition of AChE can lead to increased levels of ACh in cholinergic synapses and ultimately death. Conversely, suppressed levels of ACh may lead to memory deficits associated with Alzheimer's disease. AChE has a deep(20Å) and narrow(5Å) gorge lined with 14 aromatic residues, with its active site at the bottom of the gorge. Initially, ACh binds to the peripheral anionic site(PAS) of AChE and is funneled down the gorge to the active site by interactions between the aromatic rings of the 14 aromatic residues and the quaternary ammonium ion of ACh. At the active site, ACh is oriented for hydrolysis by interactions between the catalytic anionic ion site and the quaternary ammonium ion of ACh. The Fasciculin-II (FAS-II)toxin, a component of the East African Green Mamba snake(''Dendroaspis angusticeps'') venom, inhibits AChE by binding to the top of the active-site gorge, including residues that form the PAS; thus preventing ACh from entering the active-site gorge. The Hostos-Lincoln Academy Students Modeling A Research Topic(S.M.A.R.T) team and the Center for BioMolecular Modeling have designed and fabricated two physical models using a combination of computational molecular modeling and three-dimensional(3D) printing technology: ''Torpedo californica''(''Tc'') AChE in complex with a modeled ACh ligand and ''Tc''AChE in complex with FAS-II.
Acetylcholinesterase(AChE) is essential for hydrolysis of the neurotransmitter acetylcholine (ACh), and, therefore, for termination of impulse transmission at cholinergic synapses (Figure 2). Irreversible inhibition of AChE can result in accumulation of ACh at cholinergic synapses and, ultimately, to death. Conversely, decreased levels of ACh may result in the memory deficits associated with Alzheimer's disease<ref>PMID: 14501022</ref>. AChE has a deep (20Å) and narrow (5Å) gorge lined with 14 aromatic residues, with its active site located near the bottom of the gorge<ref>PMID: 1678899</ref>. Initially, ACh binds to the peripheral anionic site (PAS) of AChE, and is funneled down the gorge to the active site by interactions between its quaternary ammonium group and the aromatic rings of 14 aromatic amino acid residues lining the gorge. At the active site, ACh is oriented for hydrolysis by interactions between the catalytic anionic site and its quaternary ammonium group. Fasciculin-II (FAS-II), a potent polypeptide toxin present in the venom of the East African green mamba (Dendroaspis angusticeps), inhibits AChE by binding to the top of the active-site gorge, interacting tightly with residues that form the PAS; it thus prevents ACh from entering the active-site gorge<ref>PMID:8747462</ref>. The Hostos-Lincoln Academy Students Modeling A Research Topic (S.M.A.R.T) team and the Center for BioMolecular Modeling have designed and fabricated two physical models using a combination of computational molecular modeling and three-dimensional (3D) printing technology: ''Torpedo californica'' (''Tc'') AChE complexed with a modeled ACh molecule ligand, and a complex of FAS-II with ''Tc''AChE.
 
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==='''Background Information'''===
==='''Background Information'''===
[[Image:AChE-Page-Cholinergic-Synapse.jpg|thumb|alt= Alt text| Figure 2. Cholinergic Synapse |375px]]


When a nerve impulse reaches the presynaptic nerve terminal of a cholinergic synpase, it stimulates the release of the neurotransmitter, ACh (Figure 1), into the synaptic cleft. ACh diffuses across the cleft to the postsynaptic nerve terminal, where it binds reversibly to acetylcholine receptors embedded in the membrane of the postsynaptic nerve terminal. The binding of ACh to the receptors triggers a nerve impulse in the postsynaptic neuron. Finally AChE, anchored to the membrane of the postsynaptic nerve terminal (Figure 2), hydrolyzes ACh to acetate and choline, resulting in the termination of neurotransmission.
[[Image:AChE-Page-ACh_shematic.JPG|left|thumb|alt= Alt text| Figure 1. Chemical Structure of Acetylcholine |275px]]


 
Inhibition of AChE may result in various outcomes, depending on the physiological context. Toxins such as FAS-II, from the green mamba, a poisonous snake found in East Africa, inhibit AChE and ultimately lead to death. However, controlled inhibition of AChE, in patients with Alzheimer’s disease, by drugs designed for this purpose, alleviates  their symptoms, including memory loss and disorientation.
[[Image:AChE-Page-Cholinergic-Synapse.jpg|thumb|alt= Alt text| Figure 2. Cholernergic Synapse |375px]]
 
 
When a nerve impulse reaches the presynaptic nerve terminal, where it stimulates the release of the neurotransmitter, ACh, into the cholinergic synapse. ACh diffuses across the synapse to the postsynaptic nerve terminal, and binds to receptors embedded in the membrane of the  postsynaptic nerve terminal. The binding of ACh to receptors in the postsynaptic neuron re-initiates the nerve impulse. Finally AChE, anchored to the membrane of the postsynaptic nerve terminal, hydrolyzes ACh to acetate and choline resulting in the termination of the nerve  impulse at the synapse.
[[Image:AChE-Page-ACh_shematic.JPG|left|thumb|alt= Alt text| Figure 1. Chemical Structure of Acetylcholine |275px]]
Inhibition of AChE may result in different outcomes, depending on the physiological context. Toxins such as FAS-II from the East African Green Mamba snake inhibit AChE and ultimately lead to death. Conversely, reversible inhibition of AChE, in patients with Alzheimer’s disease, is an effective way to improve their symptoms, including memory loss and disorientation.


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<applet load='2ace' size='300' frame='true' align='left' scene='Sandbox_250/Ache_ach/30' caption='AChE in complex with ACh (2ace)'/>
<applet load='2ace' size='300' frame='true' align='left' scene='Sandbox_250/Ache_ach/30' caption='AChE in complex with ACh (2ace)'/>
 
<qt>file=AChE 7 26 11.m4v|width=640|height=496|autoplay=false|controller=true|loop=false</qt>
 
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<applet load='1fss' size='300' frame='true' align='left' scene='Sandbox_250/Ache_fas2/15' caption='AChE in complex with FAS-II (1fss)'/>
<applet load='1fss' size='300' frame='true' align='left' scene='Sandbox_250/Ache_fas2/15' caption='AChE in complex with FAS-II (1fss)'/>
 
<qt>file=AChE FAS 7 26 11.m4v|width=640|height=496|autoplay=false|controller=true|loop=false</qt>
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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==='''Designing Physical Models to Tell the Story of Acetylcholinesterase'''===
==='''Designing Physical Models to Tell the Story of Acetylcholinesterase'''===


Reflected in our design are two key concepts of AChE biology: the mechanism by which AChE hydrolyses ACh (the substrate traffic story), and how the Green Mamba Snake toxin, FAS-II, inhibits the hydrolysis of ACh (the inhibition story). Two physical models were designed and fabricated using a combination of computational molecular modeling and 3D printing technology: ''Tc''AChE in complex with a modeled ACh ligand, and ''Tc''AChE in complex with FAS-II. Both models were designed using the respective protein data bank (PDB) files: 2ace for the ''Tc''AChE/ACh complex and 1fss for the''Tc''AChE/FAS-II complex, and RasMol computer modeling program.  
Reflected in our design are two key concepts of AChE biology: the mechanism by which AChE hydrolyses ACh (the substrate traffic story), and how the Green Mamba Snake toxin, FAS-II, inhibits the hydrolysis of ACh (the inhibition story)<ref>PMID:18586019</ref>. Two physical models were designed and fabricated using a combination of computational molecular modeling and 3D printing technology: ''Tc''AChE in complex with a modeled ACh ligand, and ''Tc''AChE in complex with FAS-II. Both models were designed using the respective protein data bank (PDB) files: 2ace for the ''Tc''AChE/ACh complex and 1fss for the''Tc''AChE/FAS-II complex, and RasMol computer modeling program.  
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===='''Features of the Substrate Traffic Story:''a Model of'' AChE/ACh'''====
===='''Features of the Substrate Traffic Story:''a Model of'' AChE/ACh'''====
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The ''Tc''<scene name='Sandbox_250/Ache_ach/5'>AChE</scene> protein contains 537 amino acids and forms an α/β hydrolase fold. The neurotransmitter <scene name='Sandbox_250/Ache_ach/36'>ACh</scene> consists of an acytoxy group, an ethylene group and a positively charged quaternary ammonium ion.
The ''Tc''<scene name='Sandbox_250/Ache_ach/5'>AChE</scene> protein contains 537 amino acids and forms an α/β hydrolase fold. The neurotransmitter <scene name='Sandbox_250/Ache_ach/36'>ACh</scene> consists of an acytoxy group, an ethylene group and a positively charged quaternary ammonium ion.


The <scene name='Sandbox_250/Ache_ach/24'>14 aromatic residues</scene> that line the active site gorge are Tyr70, Trp84, Trp120, Tyr121, Tyr130, Trp233, Trp279, Phe288, Phe290, Phe330, Phe331, Tyr334, Trp432 and Tyr442. These aromatic residues interact with the positively charged quaternary ammonium ion of ACh by virtue of cation-π interactions to filter it down the active-site gorge to the catalytic triad.
The <scene name='Sandbox_250/Ache_ach/24'>14 aromatic residues</scene> that line the active site gorge are Tyr70, Trp84, Trp120, Tyr121, Tyr130, Trp233, Trp279, Phe288, Phe290, Phe330, Phe331, Tyr334, Trp432 and Tyr442. These aromatic residues interact with the positively charged quaternary ammonium ion of ACh by virtue of cation-π interactions to filter it down the active-site gorge to the catalytic triad (Figure 3).


The PAS includes residues <scene name='Sandbox_250/Ache_ach/11'>Tyr70, Tyr121 and Trp279</scene>. Initially, the positively charged quaternary ammonium ion of ACh is attracted to and binds to the <scene name='Sandbox_250/Ache_ach/31'>PAS of AChE</scene>, highlighted in yellow.  
The PAS includes residues <scene name='Sandbox_250/Ache_ach/11'>Tyr70, Tyr121 and Trp279</scene>. Initially, the positively charged quaternary ammonium ion of ACh is attracted to and binds to the <scene name='Sandbox_250/Ache_ach/31'>PAS of AChE</scene>, highlighted in yellow.  
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FAS-II binds to and inhibits AChE using two major mechanisms:
FAS-II binds to and inhibits AChE using two major mechanisms:


1. Long-range electrostatic complementarity: the positive lower region of FAS-II is attracted to the highly negative top region of AChE.  
1. Long-range electrostatic complementarity: the positive lower region of FAS-II is attracted to the highly negative top region of AChE (Figure 4).  


2. Amino acid specificity: residues <scene name='Sandbox_250/Ache_fas2/14'>Thr8, Arg27 and Met33</scene> are located on two of the three fingers of FAS-II. When FAS-II <scene name='Sandbox_250/Ache_fas2/18'>binds</scene> to AChE, Arg27 and Met33 interact with Trp279 part of the PAS, while Thr8 and Val34 interact with Tyr70, also part of the PAS.
2. Amino acid specificity: residues <scene name='Sandbox_250/Ache_fas2/14'>Thr8, Arg27 and Met33</scene> are located on two of the three fingers of FAS-II. When FAS-II <scene name='Sandbox_250/Ache_fas2/18'>binds</scene> to AChE, Arg27 and Met33 interact with Trp279 part of the PAS, while Thr8 and Val34 interact with Tyr70, also part of the PAS.
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3. Shape: Once bound to the PAS, two loops of FAS-II fit in to the AChE active-site gorge like a hand fits into a glove. Once this occurs, the entrance of the gorge is <scene name='Sandbox_250/Ache_fas2/13'>blocked</scene> such that acetylcholine may not enter, and therefore it will not be hydrolysed. This results in the increased levels of AChE in the cholinergic synapse, and ultimately death.
3. Shape: Once bound to the PAS, two loops of FAS-II fit in to the AChE active-site gorge like a hand fits into a glove. Once this occurs, the entrance of the gorge is <scene name='Sandbox_250/Ache_fas2/13'>blocked</scene> such that acetylcholine may not enter, and therefore it will not be hydrolysed. This results in the increased levels of AChE in the cholinergic synapse, and ultimately death.


[[Image:AChE-Page-schematic-fas.JPG|left|thumb|alt= Alt text| Figure 4. Schematic illustration of the AChE/FAS-II complex. |475px]]
[[Image:New_Schematic_AChE_Fas.JPG|left|thumb|alt= Alt text| Figure 4. AChE-fasciculin-2 complex. (a) A side view of the complex, illustrating the geometric complementarity of the two interacting proteins. AChE is presented as a yellow surface and fasciculin-2 as a blues ribbon. (b) A front view of both interacting proteins, presented separately as surfaces colored by electrostatic potential (blue is positive, white is neutral, and red is negative). To create this view, both proteins were rotated 90º compared to their position in a, AChE to the right and fasciculin to the left. The electrostatic compatibility between the two proteins is clear; The positively charged part of fasciculin matches the entrance to AChE's binding site, which is negatively charged <ref>Kessel A and Ben-Tal N (Dec. 2010) Introduction to Proteins: Structure, Function, and Motion. Chapman & Hall/CRC Mathematical & Computational Biology. ISBN: 9781439810712</ref>.|500px]]


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==='''References'''===
==='''References'''===
<references/>


1. Sussman, Joel L., Harel M., Frolow, F., Oefner, Christian, Goldman, Adrian, Toker, Lilly, Silman, Israel (2006). Atomic Structure of Acetylcholinesterase form Torpedo californica:  A Prototypic Acetylcholine-Binding Protein in Science 253, 872-879.
2. Goodsell, David, “Acetylcholinesterase.”  Protein Data Bank:  Molecule of the Month. Web. June 2004.
3. Silman, Israel, Sussman, Joel L. (2008).  Acetylcholinesterase:  How is Structure Related to Function? In Chemico-Biological Interactions 175(3-10).
4. Harel, Michal, Kleywegt, Gerrad J., Ravelli, Raimond B.G., Silman, Isreal, Sussman, Joel (1995).  Crystal Structure of an Acetylcholinesterase-Fasciculin Complex:  Interaction of a Three-Fingered Toxin From Snake Venom With Its Target in Structure 3(12), 1355-1366.
5. Greenblatt, Harry M., Dvir, Hay,Silman, Isreal, Sussman, Joel L. (2002). Acetylcholinesterase:  A Multidaceted Target for Strucutre-Based Drug Design of Anitcholinesterase Agents for the Treatment of Alzheimer’s Disease in Journal of Molecular Neuroscience 20, 369-383.
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7. Michal Harel, Weizmann Institute of Science
7. Michal Harel, Weizmann Institute of Science


8. Malcolm Twist
8. Natural Sciences Department,Hostos Community College, Bronx, NY
 
9. Malcolm Twist
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Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

Joel L. Sussman, Allison Granberry, Jaime Prilusky
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