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Molecular Dynamics Simulations and Docking Studies of AChBP and the Ligand
Binding Domain of α7 nAChR
Shiva Amiri
JC 20-04-2005
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1. Simulation studies of AChBP with Nicotine, Carbamylcholine, and HEPES as ligands
> also one simulation of the ligand binding domain of α7 nAChR
2. Docking studies of α7 nAChR with Nicotine, Imidacloprid (an insecticide), and Acetylcholine (ACh)
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nAChR
a ligand gated ion channel (LGIC) found in central and peripheral nervous system
endogenous ligand is acetylcholine (ACh) but reactive to many compounds such as nicotine, alcohol, and toxins
mutations lead to various diseases such as epilepsy, myasthenic syndromes, etc.
implicated in Alzheimer’s disease and Parkinson’s disease (not well understood)
mediates nicotine addiction
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4Å structure of nAChR
Ligand binding domain (LB) core of 10 β-strands, forming a β-sandwich an N-terminal α-helix, two short 310 helices
Transmembrane domain (TM) 4 α-helices in each subunit (M1-M4)
Intracellular domain (IC) α-helical, some residues still missing
Unwin, Journal of Molecular Biology, March, 2005
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AChBP
AChBP – from Lymnaea stagnalis, high homology with the ligand binding domain of ligand gated ion channels (LGICs) i.e. nAChR, GABA, Glycine, 5-HT3
> Highest sequence identity with homomeric nAChR
Celie et al., Neuron, March 2004
AChBP with HEPES (1UX2) – 2.1 Å
AChBP with Carbamylcholine (1UV6) – 2.5 Å
AChBP with Nicotine (1UW6) – 2.2 Å
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List of simulations
apo AChBP (1UX2) 10 ns
apo AChBP (1UW6) 10 ns
apo AChBP (1UV6) 10 ns
apo α7 nAChR LB domain (model)
10 ns
AChBP (1UX2) with HEPES
10 ns
AChBP (1UW6) with Nicotine
10 ns
AChBP (1UV6) with Carbamylcholine
10 ns
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The Ligands …
Nicotine
Carbamylcholine
HEPES
ACh derivative, 10-fold less binding affinity for AChBP compared to ACh
successful binding under crystallization conditions
Very high affinity for both nAChR and AChBP
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Making the topologies…
InsightII was used for protonating the ligands and Spartan was used to get
the charges Further details on making a topology on
http://indigo1.biop.ox.ac.uk/wiki/index.php/Making_a_topology_file_-_a_quick_guide
For HEPES, I used PRODRG2.5 (beta), it gives GROMOS96 topologies> have to check the topologies produced by this server…there are
some bugs A 1 ns simulation in water was run on each ligand after making its topology
before including it with the protein
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apo AChBP (1UV6)
Crystal structure had two Carbamylcholines bound in binding site in two adjacent subunits
Higher covariance near TM domain, in subunits where the ligands were bound in crystal structure
GNM run showing highest flexbility of ligand binding region, as well as the bottom where the LB domain joins the TM domain
http://s12-ap550.biop.ox.ac.uk:8078/dynamite_html/index.html
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AChBP + nicotine
rmsf plot (rmsf values as B-factor values)
the region nearest the TM domain, and the ligand binding site are most flexible as well as the very top of the receptor
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AChBP + Nicotine: PCA
Covariance line plot (70%) (top view)
Heavier covariance in two of the 5 subunits
Covariance line plot (80%) (side view)
Heavier covariance at the very top and the very bottom of subunits, where it meets the TM domain
porcupine plot of the first eigenvector (top view)
larger eigenvalues in two of the 5 subunits
http://s12-ap550.biop.ox.ac.uk:8078/dynamite_html/index.html
agrees with simulations of AChBP bound to Ach where only 2 ACh molecules are required to keep AChBP in ligand bound state rather than 5 (Gao et al., J. Biol. Chem, 2005)
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The Binding Site
Ligands bind in the interface between two subunits
> the principal (+) side composed of loops A, B, C and the complementary side (-) composed of loops D and E
Ligand is completely buried in the protein
Brejc et. al., Nature, May 2001
Ligand sitting behind the C-loop of the principal side of the receptor
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Nicotine binding
Hydrophobic interactions with surrounding residues
Hydrogen bonding with Ser349, Trp350
it is thought that the bridging water molecules with Leu515 and Met527 contribute significantly to the binding of NCT
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Figure showing the hydrophobic interactions mostly exist between Trp350 and Nicotine
Also between cys395 and Nicotine
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‘Breathing’ motion Gain of symmetry upon ligand
binding?
First principal component Nicotine in binding pocket
Nicotine is stationery at its protonated N
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Next …
Docking of ligands every x frames to look at binding behaviour throughout the simulation (using AUTODOCK)
Nicotine docked onto the binding pocked of AChBP
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Docking studies of α7 nAChR
Some α7 background:
Homopentameric cationic channel Found in central nervous system Implicated in learning disabilities, Parkinson’s, Alzheimer’s, alcoholism, and
nicotine addiction
Docking:
The ligand binding domain is used for the docking studies with AUTODOCK Modelled on new AChBP HEPES bound structure (2.1 Å) (Celie et al.,
Neuron, March 2004) using MODELLER Nicotine (NCT), Acetylcholine (ACh), and Imidacloprid (IMI) used as ligands
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Leu118
Leu118 is believed to be involved in the selectivity and binding of agonists
Docking carried out with wild type (WT), and L118D, L118E, L118K, L118R mutations for all three ligands
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WT and Mutations for Nicotine
Binding energies for mutations (lowest to highest): E, D, R, K
all 50 WT docks in the exact same position in binding site
L118R
L118EL118D
L118K
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ACh WT
Lowest energy dock
Smaller molecule, may be able to bind in different orientations
Simulation studies of ACh with α7 nAChR reveal very mobile behaviour of ACh in binding pocket (Henchman et al., Biophys. J., April 2005)
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ACh and NCT binding
The lowest energy, highest ranked docks of NCT and ACh puts the ammomium group in the same position
NCT bound ACh bound NCT and ACh superimposed
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Further docking
Fighting with Imidacloprid docks… More ACh docking to look for a
more clear pattern Using the 4Å Torpedo marmorata
(Unwin, Journal of Molecular Biology, March, 2005) structure for docks to compare binding sites and modes of ligand binding
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Summary + Future Directions
Simulations Simulations show highest covariance and flexibility near the TM domain, in
ligand binding site, and at the very top of the receptor Higher covariance in subunits with bound ligand, even in APO simulations First eigenvector shows ‘breathing motion’ in agreement with Henchman’s
data Further analysis on individual subunits, binding site, ligand contacts and
behaviour needs to be done
Docking Mutations cause incorrect binding orientations of nicotine ACh … multiple binding modes? IMI in progress Heteropentameric EM structure will be used for further docking and
comparison of different binding sites
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L118D L118E
L118K L118R