1. Molecular Modelling Studies of the Nicotinic Acetylcholine Receptor
Shiva Amiri
Professor Mark S. P. Sansom and Dr. Philip C. Biggin
D. Phil. Symposium, October 6, 2005

2. The Nicotinic Acetylcholine Receptor (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
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

3. Computational methods to study membrane proteins
There are very few crystal structures available for membrane proteins
can build structures and use them to perform a range of studies such as electrostatics, pore profiling, ligand docking, Molecular Dynamics simulations etc.
Studying the movement of proteins to gain insight into their function
various methods of using a structure to look at the dynamics of the protein
Docking of ligands onto receptors
drug design

4. Generating Structures
Scoring criteria
chosen {θ, z}
theta (radians)
z translation (Å) x
O. Beckstein, K. Tai
Possible gate region
Amiri et al., Mol. Mem. Biol, 2005

5. Motion Analysis Atomistic Molecular Dynamics Simulations with GROMACS
Coarse-grain Grouping of atoms
Gaussian Network Model (GNM) Assessing the flexibility of structures depending on the number of neighbouring residues
CONCOORD
Generating random structures from a given structure within distant constraints
In-house methods Grouping data to simplify MD output
Ligand Docking
Docking Nicotine and other ligands onto various frames of trajectories
Water
Looking at the behaviour of water in the binding pocket

6. Coarse-grain methods (1)
Gaussian Network Model (GNM)
A coarse-grained method to approximate fluctuations of residues based on the number of neighbours within a cut-off radius
Information on the flexibility of the receptor, may outline functionally important regions of the protein
ligand binding site

7. Coarse-grain methods (2)
Porcupine plot showing the nAChR’s two domains rotating in opposite directions. Suggests motions that could be involved in the gating mechanism
(Barrett et al., 2004)
Covariance matrix showing which part of the protein moves together. The red parts show highest covariance and the blue indicates negative covariance (move in opposite directions)
SUB1
SUB3
SUB4
SUB5
SUB2
CONCOORD
Generates n number of structures that meet distance constraints
very quick: one run takes a few minutes
Output used in Principle Component Analysis (PCA) to describe major modes of motion

8. Molecular Dynamics Simulations
Covariance lines show which sections of the receptor are moving together
(Barrett et al., 2004)
A method to study conformational changes at an atomistic level
MD of ligand binding domain of nAChR homologue, AChBP (Celie et al., 2004)
several simulations are being carried out for AChBP:
i) non-liganded simulations
ii) with various ligands: nicotine, carbamylcholine, HEPES
One nanosecond takes ~ 5 days for this system
Actual gating of this receptor happens on a millisecond time-scale

9. Molecular Dynamics Simulations continued …
A simulation of AChBP with Nicotine in all 5 binding sites.

10. The Binding Pocket
MET 115
TRP 144
LEU 103
THR 145
CYS 188
CYS 189
The important residues in the binding pocket are shown. These residues are thought to have key interactions with the ligand.
Two subunits of the nAChR are shown with Nicotine inside the binding pocket
Studying structural changes which occur in the binding pocket to better understand how binding of a ligand results in the functioning of the ion channel
looking at distances, dihedrals of surrounding residues, and the behaviour of water in the binding pocket

11. Water in the Binding Pocket
Zone 2
Zone 1
Zone 4
Zone 3
Zone 5
Bridging waters form hydrogen bonds between the ligand and surrounding residues (shown using Ligplot)
Water seems to play an important role in ligand binding. There are various zones in the binding pocket where waters are frequently present

12. Water in the Binding Pocket
Water molecules which remain in their position in the Binding Pocket

13. Docking Nicotine Carbamylcholine HEPES
Docking various ligands such as nicotine, acetylcholine, imidacloprid (an insecticide) onto AChBP and the α7 nAChR to look at possible binding modes
An automated docking program docks ligands onto hundreds of frames from a trajectory
Nicotine docked onto the AChBP binding site
Nicotine
Carbamylcholine
HEPES

14. Results Structure Generation:
Developed a method to generate protein structures from their separate domains
Molecular Dynamics:
MD studies of AChBP with Nicotine, Carbamylcholine, and HEPES
Studying the role of water in the binding pocket
MD of α7 nAChR mutants
Coarse-graining:
GNM
CONCOORD
Grouping of information from MD trajectories
Automated Docking:
Automated docking of ligands (Nicotine, acetylcholine, carbamylcholine, insecticides) onto AChBP and nAChR (and its mutants) along a trajectory

15. Thanks to… Prof. Mark S. P. Sansom Dr. Philip C. Biggin
Dr. Alessandro Grottesi
Dr. Kaihsu Tai
Dr. Zara Sands
Dr. Oliver Beckstein
Dr. Daniele Bemporad
Dr. Jorge Pikunic
Dr. Andy Hung
Dr. Shozeb Haider
Dr. Syma Khalid
Dr. Pete Bond
Dr. Kia Balali-Mood
Dr. Hiunji Kim
Dr. Bing Wu
Sundeep Deol
Yalini Pathy
Jonathan Cuthbertson
Jennifer Johnston
Katherine Cox
Robert D’Rozario
Jeff Campbell
Loredana Vaccaro
John Holyoake
Tony Ivetac
Samantha Kaye
Sylvanna Ho
Benjamin Hall
Emi Psachoulia
Chze Ling Wee

17. Future Work
Further investigation of the role of water in the binding pocket
Analysis of simulations of α7 nAChR mutants and docking along their trajectories
Development of further methods for understanding the motion of proteins from the limited structural data available
Combining coarse-grained and MD data…. i.e. Running GNM on various frames of a trajectory

18. Coarse-grain methods (3)
Grouping eigenvectors
simplifying Molecular dynamics data by grouping the eigenvectors from the resulting trajectory.

19. The Binding Pocket
MET 115
TRP 144
LEU 103
THR 145
CYS 188
CYS 189
Studying structural changes which occur in the binding pocket to better understand how binding of a ligand results in the functioning of the ion channel
looking at distances, dihedrals of surrounding residues, and the behaviour of water in the binding pocket
The important residues in the binding pocket are shown. These residues have been shown to interact with the ligand.

21. Using computational techniques to study the most flexible regions of the nAChR. These residues could play a key role in the gating of the receptor. Red shows the most flexibility, with blue showing least movement.