Sandbox 250

A Physical Model of Acetylcholinesterase in Complex with Acetylcholine and Fasciculin-IIA Physical Model of Acetylcholinesterase in Complex with Acetylcholine and Fasciculin-II

Students: Mary Acheampong. Daviana Dueno, Bobby Glover, Alafia Henry, Randol Mata, Marisa VanBrakle. Teacher: Allison Granberry Mentors: Joel Sussman, Weissman Institule of Science, and Lars Westblade, touro College of Pharmacy.

AbstractAbstract

Acetylcholinesterase(AChE) is essential for the hydrolysis of the neurotransmitter acetylcholine(ACh) in cholinergic synapses. Irreversible inhibition of AChE can lead to increased levels of ACh and ultimately death. Conversely, suppressed levels of ACh may lead to memory deficits associated with Alzheimer's disease. AChE has a deep(20A) and narrow(5A) 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, from the East African Green Mamba snake(Dendroaspis angusticeps) venom, inhibits AChE by binding to the top of the active-site gorge, and thus preventing ACh from entering into it. The Hostos-Lincoln Academy SMART(Students Modeling A Research Topic) team and MSOE have designed and made two physical models by three-dimensional(3D) printing technology: Torpedo californica(Tc) AChE in complex with a modeled ACh ligand and TcAChE in complex with FAS-II.

Designing a Physical Model to Tell the Story of AcetylcholinesteraseDesigning a Physical Model to Tell the Story of Acetylcholinesterase

Reflected in our design are two key concepts of AChE: the biochemistry of how the ACh overcomes the depth of the active site gorge before hydrolysis can occur, and how a toxin inhibits the substrate from finding the active site.Two physical models were designed and made by 3-dimensional printing technology: Torpedo californica (Tc) AChE in complex with a modeled ACh ligand, and Tc AChE in complex with FAS-II. Both models were based on protein data bank (PDB) files, and Rasmol computer modeling program. PDB files included PDB entry code 2ace for the TcAChE/ACh complex, and PDB entry code 1fss for the Tc AChE/FAS-II complex.

spinqualitylabelsall models AChE/ACh Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Features of the Substrate Traffic Story: AChE/AChFeatures of the Substrate Traffic Story: AChE/ACh

is an alpha/beta hydrolase fold with an amino acid sequence of 4-535. consists of an acytoxy group, ethylene group and a quaternary ammonium ion.

The that line the active site gorge are tyr70, trp84, trp120, tyr121, tyr130, trp233, trp279, phe288, phe290, phe330, phe331, tyr334, trp432 and tyr442.

The Peripheral Anionic Site(PAS) includes . Initially, the positively charged quaternary ammonium ion of ACh is attracted to and binds to the , highlighted in yellow.

The Catalytic Anionic Site(CAS) includes . The , highlighted in red, holds ACh in the optimal position for hydrolysis by interacting with the quaternary ammonium ion of ACh.

The active site includes three residues: . The , highlithed in blue, is responsible for the hydrolysis of ACh into acetate and choline.

spinqualitylabelsall models AChE/FAS-II Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Features of the Inhibition StoryFeatures of the Inhibition Story

FAS-II is a 61-residue polypeptide with 4 beta sheets forming three loops or fingers.

FAS-II is attracted to AChE by a number of mechanisms:

1. Amino acid specificity: Thr8, Met33, Arg27 and Val34 are located on two of the three fingers of the FAS-II. When FAS-II binds to AChE, Arg27 and Met33 interact with Trp279 of the Peripheral Anionic Site, while Val34 and Thr8 interact with Tyr70 of the Peripheral Anionic Site.

2. Shape: Once bound to the PAS, two loops of FAS-II fit in to the active-site gorge like a hand fits into a glove. Once this interaction occurs, the entrance of the gorge is blocked such that acetylcholine may not enter.

ReferencesReferences

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.

AcknowledgementsAcknowledgements

1. Bill & Melinda Gates Foundation

2. Howard Hughes Medical Institue Pre-College Program

3. Center for BioMolecular Modeling, Milwaukee School of Engineering

4. The Rockefeller University Center for Clinical and Translational Science

5. The Rockefeller University SMART Team Program

6. The Rockefeller University Science Outreach Program

7. Malcolm Twist