Molecular%20Dynamics%20Simulations%20and%20Docking%20Studies%20of%20AChBP%20and%20the%20Ligand%20Binding%20Domain%20of%20a7%20nAChR

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Molecular Dynamics Simulations and Docking
Studies of AChBP and the Ligand Binding Domain of
a7 nAChR

• Shiva Amiri
• JC 20-04-2005

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• Simulation studies of AChBP with Nicotine,
  Carbamylcholine, and HEPES as ligands
• gt also one simulation of the ligand binding
  domain of a7 nAChR
• Docking studies of a7 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 Alzheimers disease and Parkinsons
  disease (not well understood)
• mediates nicotine addiction

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4Å structure of nAChR

• Ligand binding domain (LB)
• core of 10 ß-strands, forming a ß-sandwich
• an N-terminal a-helix, two short 310 helices

• Transmembrane domain (TM)
• 4 a-helices in each subunit (M1-M4)

• Intracellular domain (IC)
• a-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
• gt Highest sequence identity with homomeric nAChR

Celie et al., Neuron, March 2004
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List of simulations
apo AChBP (1UX2) 10 ns
apo AChBP (1UW6) 10 ns
apo AChBP (1UV6) 10 ns
apo a7 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
Very high affinity for both nAChR and AChBP

• Nicotine
• Carbamylcholine
• HEPES

ACh derivative, 10-fold less binding affinity for
AChBP compared to ACh
successful binding under crystallization
conditions
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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/M
  aking_a_topology_file_-_a_quick_guide
• For HEPES, I used PRODRG2.5 (beta), it gives
  GROMOS96 topologies
• gt have to check the topologies produced by this
  serverthere 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

• GNM run showing highest flexbility of ligand
  binding region, as well as the bottom where the
  LB domain joins the TM domain

• Higher covariance near TM domain, in subunits
  where the ligands were bound in crystal structure

http//s12-ap550.biop.ox.ac.uk8078/dynamite_html/
index.htm l
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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

• porcupine plot of the first eigenvector (top
  view)
• larger eigenvalues 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

• Covariance line plot (70) (top view)
• Heavier covariance in two of the 5 subunits

• 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)

http//s12-ap550.biop.ox.ac.uk8078/dynamite_html/
index.htm l
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The Binding Site

• Ligands bind in the interface between two
  subunits
• gt 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

Ligand sitting behind the C-loop of the principal
side of the receptor
Brejc et. al., Nature, May 2001
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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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First principal component
Nicotine in binding pocket

• Breathing motion
• Gain of symmetry upon ligand binding?

• 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 a7 nAChR

• Some a7 background
• Homopentameric cationic channel
• Found in central nervous system
• Implicated in learning disabilities, Parkinsons,
  Alzheimers, 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

• all 50 WT docks in the exact same position in
  binding site

• Binding energies for mutations (lowest to
  highest) E, D, R, K

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ACh WT
Lowest energy dock

• Smaller molecule, may be able to bind in
  different orientations
• Simulation studies of ACh with a7 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
NCT and ACh superimposed
NCT bound
ACh bound

• The lowest energy, highest ranked docks of NCT
  and ACh puts the ammomium group in the same
  position

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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 Henchmans 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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