Molecular Dynamics Simulations and Docking Studies of AChBP and the Ligand

Binding Domain of α7 nAChR

Shiva Amiri

JC 20-04-2005

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)

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

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

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

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

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

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

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

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

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)

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

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

Figure showing the hydrophobic interactions mostly exist between Trp350 and Nicotine

Also between cys395 and Nicotine

‘Breathing’ motion Gain of symmetry upon ligand

binding?

First principal component Nicotine in binding pocket

Nicotine is stationery at its protonated N

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

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

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

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

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)

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

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

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

L118D L118E

L118K L118R