Proteopedia - User contributions [en]

Journal:BAMBEd:Acetylcholinesterase: Substrate Traffic and Inhibition

2011-11-03T19:27:59Z

Mary Acheampong:

=='''Acetylcholinesterase: A Story of Substrate Traffic and Inhibition'''==

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==='''Introduction'''===

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'''===
[[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.

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==='''Models of AChE'''===
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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)'/>
<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)'/>
<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'''===

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'''====
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<applet load='2ace' size='500' frame='true' align='left' scene='Sandbox_250/Ache_ach/1' caption='AChE/ACh'/>
[[Image:AChE-Page-schematic-gorge.jpg|thumb|alt= Alt text| Figure 3. Schematic illustration of AChE. |525px]]

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 (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 Catalytic Anionic Site (CAS) includes residues <scene name='Sandbox_250/Ache_ach/18'>Trp84 and Phe330</scene>. The
<scene name='Sandbox_250/Ache_ach/34'>CAS</scene>, highlighted in red, holds ACh in the optimal position for hydrolysis by interacting with the quaternary ammonium ion of ACh.

The AChE active site includes three residues that form a catalytic triad: <scene name='Sandbox_250/Ache_ach/20'>Ser200, Glu327, and His440</scene>. The <scene name='Sandbox_250/Ache_ach/33'>Catalytic Triad</scene>, highlighted in blue, is responsible for the hydrolysis of ACh into acetate and choline.

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===='''Features of the Inhibition Story: a Model of AChE/FAS-II'''====
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<applet load='1fss' size='480' frame='true' align='right' scene='Sandbox_250/Ache_fas2/6' caption='AChE/FAS-II' />

The Green Mamba snake toxin, <scene name='Sandbox_250/Ache_fas2/9'>FAS-II</scene>, is a 61-residue protein that folds into 4β sheets, with 3 of the 4β sheets forming loops, or fingers.

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 (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.

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: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/>

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==='''Acknowledgements'''===

1. Howard Hughes Medical Institue Pre-College Program

2. Center for BioMolecular Modeling, Milwaukee School of Engineering

3. The Rockefeller University Center for Clinical and Translational Science

4. The Rockefeller University S.M.A.R.T Team Program

5. The Rockefeller University Science Outreach Program

6. Touro College of Pharmacy

7. Michal Harel, Weizmann Institute of Science

8. Natural Sciences Department,Hostos Community College, Bronx, NY

9. Malcolm Twist
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Journal:BAMBEd:Acetylcholinesterase: Substrate Traffic and Inhibition

2011-11-03T19:26:55Z

Mary Acheampong:

Sandbox 121

2011-10-30T15:34:29Z

Mary Acheampong:

=='''Modeling of Beta-2 Adrenergic Receptor: Ligand Binding and Activation'''==

Students: Mary Acheampong, Kavita Bhikhi, Daviana Dueno, Bobby Glover, Lachoy Harris, Alafia Henry, Randol Mata, and Marisa Vanbrakle, Hostos-Lincoln Academy of Science

Teacher: Allison Granberry, Hostos-Lincoln Academy of Science

Mentors: Thijs Beuming, Schrodinger, Haregewein Assefa,Touro College of Pharmacy

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==='''Introduction'''===
The Beta-2 Adrenergic Receptor (B₂AR) is a G-protein coupled receptor (GPCR) which, when stimulated by a catecholamine, causes the relaxation of various smooth muscles, and the production of glucose by glycogenolysis and gluconeogenesis. Pharmaceuticals acting through B2AR are important for treating asthma, chronic obstructive pulmonary disease (COPD), and premature labor. The structure of B2AR consists of 7-transmembrane domains, connected by three extracellular loops and three intracellular loops. At the base of the extracellular loops, buried within the transmembrane helices, there is a predominately hydrophobic binding pocket with several crucial polar residues that interact with ligands. Interestingly, certain polar interactions appear to play a role in the conversion of the receptor from an active to an inactive state. Recent crystallography of B2AR has revealed that the active state, relative to the inactive state, shows only minor changes in the binding pocket, whereas critical shifts occur at the cytoplasmic face. These conformational changes lead to a dissociation of the G-protein from the receptor, which then initiates a signaling cascade. The Hostos-Lincoln Academy SMART team (Students Modeling A Research Topic) modeled ligands in complex with B2AR using 3D printing technology. Supported by grants from the HHMI Precollege Program and the Camille and Henry Dreyfus Foundation.

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==='''Background Information'''===
[[Image:B2AR-Adernergic synapse.JPG|thumb|alt= Alt text| Adernergic Synapse |400px]]

Adrenergic receptors are involved in activation of the sympathetic nervous system following sudden external stimuli. After arrival of a nerve impulse, the neurotransmitter norepinephrine (NE) is released from the presynaptic terminal of the sympathetic neuron. NE is a tyrosine derived [[Image:B2AR-Norepinephrine.JPG|left|thumb|alt= Alt text| Norephrine |300px]] catecholamine containing an amino-hydroxyethyl and a catechol group.
NE binds to adrenergic receptors embedded in the postsynaptic effector cell membrane.
Following binding of NE, to either alpha or beta receptors, conformational changes in the receptor lead to a disassociation of the G protein from the cytoplasmic face of the receptor which activates a second messenger, initiating a signaling cascade.
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<applet load='3P0G' size='300' frame='true' align='right' scene='Sandbox_121/B2ar_struc/12' caption='B₂AR Active'/>

==='''Structure of Beta 2 Adrenergic Receptor'''===

B2AR is a single chain that crosses the lipid membrane 7 times from the extracellular to cytoplasmic surface.
There are 3 extracellular loops and 3 intracellular loops. The <scene name='Sandbox_121/B2ar_struc/9'>binding pocket</scene> is located to the center of the extracellular surface.

When B2AR is activated, the G-protein disassociates. A surrogate nanobody, <scene name='Sandbox_121/B2ar_struc/13'>Nb80</scene>, that is a camelid antibody produced to mimic the G-protein needed for an active state of the B2AR.

Active B₂AR in complex with BI-167107: <scene name='Sandbox_121/B2ar_struc/7'>Polar</scene> and <scene name='Sandbox_121/B2ar_struc/8'>Hydrophobic</scene> Interactions

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==='''Ligands'''===
To understand activation of β₂AR by various ligands, it is first essential to define the term baseline activity. In the absence of a ligand within the binding pocket, there is some basal activity between the receptor and its signaling pathway. This activity is not considered an active state of the receptor but simply the baseline activity of the receptor. There are two extremes to the activity of the receptor that can be seen with binding of either an inverse agonist or an agonist. The inverse agonist reduces the activity to the level below that of the basal activity of the receptor whereas the agonist activates the receptor to its maximum. A ligand that is an antagonist actually has no effect on the basal activity of the receptor. An antagonist simply sterically blocks the receptor so that no other ligand can bind. Their activity would be considered baseline. Antagonists for the adrenergic receptors are commonly called Beta Blockers. Although Beta Blockers are not prescribed for for their β₂AR blocking activity, they are frequently prescribed for their action on the Beta-1 Adrenergic Receptors in people with cardiovascular disorders such as hypertension and angina. A number of selective β₂AR agonists are used for the treatment of asthma and chronic obstructive pulmonary disease (COPD).

{| border="1" cellpadding="2"
! scope="col" width="50" | Name
! scope="col" width="150" | Description
! scope="col" width="250" | Chemical Structure
! scope="col" width="250" | Photo
|-
| Isoproterenol || Isoproterenol is an agonist that is structurally similar to NE and readily binds to β₂AR with high affinity. Isoproterenol contains an isopropyl amino group and a catechol group. || [[Image:B2AR-Isoproterenol-edited-structure.jpg|thumb|center|alt= Alt text| |300px]] || [[Image:Isoproterenol-Picture.JPG|thumb|center|alt= Alt text| |250px]]
|-
| BI-167107 || The active state of β₂AR was crystallized using BI-1671071 . Although it is not a catecholamine, it is a full agonist. || [[Image:B2AR-BI-167107-Structure.JPG|thumb|center|alt= Alt text| |300px]] || [[Image:B2AR-BI-167107-Picture.JPG|thumb|center|alt= Alt text| |250px]]
|-
| Carazolol || Carazolol is an inverse agonist designed to inhibit β₂AR. Carazolol contains a propylamino and a carbazole group. || [[Image:B2AR-Carazolol-Structure-2.JPG|thumb|center|alt= Alt text| |250px]] || [[Image:B2AR-Carazolol-Picture.JPG|thumb|center|alt= Alt text| |250px]]
|}

===='''Comparison of the Inverse Agonist and the Agonist'''====
Notable differences between carazolol and both isoproterenol and the natural agonist norepinephrine are that:
(i) Carazolol lacks the hydroxyl groups thought to be necessary for the activation of β₂AR.
(ii)The side chain of carazolol is two atoms (one carbon and one oxygen) longer in length from the amino group to the carbazole moiety.
These are common characteristics of β₂AR antagonists.

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[[Image:B2AR_Binding_Pocket_PolarInt_Act_In.JPG|right|thumb|alt= Alt text| The binding mode of isoproterenol and carazolol in B2AR. Hydrophobic residues are displayed in yellow. Polar interactions are displayed with residues in cyan, oxygen in red, and hydrogen in white. (a) A model of B2AR in its active state in complex with isoproterenol.
(b) B2AR in its inactive state in complex with carazolol. |450px]]
==='''Ligand Binding'''===

Ligands share several key interactions in the binding pocket including:

(i)Polar interactions between:

The amine and Asp113 in TM3, Asn312 in TM7, and Tyr316 in TM7.

Hydroxyls and other h-bond donors and Ser207 in TM5, Ser203 in TM5, and Asn293 in TM6.

(ii) Hydrophobic interaction between ligand and Val117 in TM3, Phe193
in ECL2, Phe289 in TM6, and Phe290 inTM6.

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==='''Conformational Change'''===
<Structure load='Morph_2rh1_on_3p0g_all_atoms.pdb' size='500' frame='true' align='left' caption='Conformational Changes in B2AR from Inactive State(2rh1) to Active State(3p0g)' scene='Sandbox_254/B2ar_morph_ser_glu_arg/32'/>‎
When an agonist is in the <scene name='Sandbox_254/B2ar_morph_ser_glu_arg/23'>binding pocket</scene> a 2.1Å inward movement of TM5 at Ser207 is observed. This bulge at ser207 allows for a hydrogen bond between the ligand and the receptor. This interaction appears to be a key event in activation.

[[Image:B2AR-Binding_Pocket_OH.JPG|thumb|right|alt= Alt text| Models of isoproternol binding to two B2AR structures. (a) Inactive B2AR: 4.78Å distance between the catechol-OH of the ligand and Ser207 of TM5 is too large for a H-bond. (b) Active B2AR: A hydrogen bond distance of 2.17Å between the catechol-OH of the ligand and Ser207 on TM5 is shown. |450px]]
[[Image:B2AR-Binding_Pocket_clash.JPG|thumb|right|alt= Alt text| Model of carazolol binding to B2AR structure. (a) Active B2AR: there is a steric clash between the ligand and Ser207 of TM5. (b)Inactive B2AR: carazolol in B2AR fits perfectly and blocks the agonist from entering the binding pocket. |450px]]
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After the agonist binds, there is a rearrangement of interactions between residues located beneath the binding pocket that contributes to a rotation and outward movement of TM6 at Phe282. This change is associated with the breaking of the ionic lock between Glu268 in TM6 and Arg131 in TM3, resulting in an 11.4Å outward movement of the helix at the cytoplasmic face.

==='''Molecular Morph'''===
The coordinates for molecular morphs between inactive state of B2AR (2rh1) and active state (3p0g) were generated using iPyMOL and eMovie (http://www.weizmann.ac.il/ISPC/eMovie.html). Morphs, a series of 10 linear interpolations between a starting and finishing model, are useful when viewing the transition of a conformational change. This model of B2AR using morphs should not be thought of as precise animation of conformational changes upon activation but rather as a comparison of the inactive state to the active state.

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==='''Reference'''===
1. Vadim Cherezov, Daniel M. Rosenbaum, Michael A. Hanson, Søren G. F. Rasmussen, Foon Sun Thian, Tong Sun Kobilka, Hee-Jung Choi, Peter Kuhn, William I. Weis, Brian K. Kobilka, Raymond C. Stevens (2007). High Resolution Crystal Structure of an Engineered Human B2-Adrenergic G Protein- Coupled Receptor Science 318, 1258-1265.

2.Søren G. F. Rasmussen, Hee-Jung Choi, Juan Jose Fung, Els Pardon, Paola Casarosa, Pil Seok Chae, Brian T. DeVree, Daniel M. Rosenbaum, Foon Sun Thian, Tong Sun Kobilka, Andreas Schnapp, Ingo Konetzki, Roger K. Sunahara,Samuel H. Gellman, Alexander Pautsch, Jan Steyaert, William I. Weis & Brian K. Kobilka (2011). Structure of a nanobody-stabilized active state of the B2 adrenoceptor Nature 469, 175-180.

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==='''Acknowledgements'''===
Camille and Henry Dreyfus Foundation,
The Rockefeller University Center for Clinical and Translational Science,
The Rockefeller University Science Outreach Program,
Howard Hughes Medical Institute Pre-college Program,
Center for BioMolecular Modeling, Milwaukee School of Engineering,
The David A. Cofrin Center for Biomedical Information,
in the HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College,

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