Transcript D. Phil. Symposium October 5, 2005

Molecular Modelling of the Nicotinic
Acetylcholine Receptor and Related Proteins
Shiva Amiri
Professor Mark S. P. Sansom and Dr. Philip C. Biggin
D. Phil. Symposium, October 6, 2005
The Nicotinic Acetylcholine Receptor
(nAChR)

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


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
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
Computational methods to study membrane
proteins

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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
Generating Structures
Scoring criteria
chosen {θ, z}
theta
(radians)
x
z translation (Å)
O. Beckstein, K. Tai
Possible
gate
region
Amiri et al., Mol. Mem. Biol, 2005
Motion Analysis
Atomistic
Coarse-grain
Molecular Dynamics
Simulations with
GROMACS
Ligand Docking
Water
Docking Nicotine
and other ligands
onto various frames
of trajectories
Looking at
the behaviour
of water in
the binding
pocket
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
Eigenvectors to
simplify MD data
Coarse-grain methods (1)
Gaussian Network Model
(GNM)
ligand binding site
 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
140
120
B-values
100
80
60
40
20
0
1
101
201
301
401
501
601
701
801
901 1001 1101 1201 1301 1401 1501 1601
number of residues
Coarse-grain methods (2)
CONCOORD
SUB1
 Generates n number of structures that
meet distance constraints
SUB2
 very quick: one run takes a few
SUB3
minutes
SUB4
 Output used in Principle Component
Analysis (PCA) to describe major modes
of motion
SUB5
SUB1
Porcupine plot showing the
nAChR’s two domains rotating in
opposite directions. Suggests
motions that could be involved in
the gating mechanism
SUB2
SUB3
SUB4
SUB5
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)
http://s12-ap550.biop.ox.ac.uk:8078/dynamite_html/index.html
(Barrett et al., 2004)
Molecular Dynamics Simulations
 A method to study conformational
changes at an atomistic level
 MD of ligand binding region of
AChBP (nAChR homologue) (Celie
et al., 2004)
 several simulations are being
carried out for nAChR:
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
Covariance lines show which
sections of the receptor are
moving together
http://s12-ap550.biop.ox.ac.uk:8078/dynamite_html/index.html (Barrett et al., 2004)
Molecular Dynamics Simulations continued …
A simulation of AChBP
(nAChR ligand binding
domain homologue) with
Nicotine in all 5 binding
pockets.
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
TRP 144
LEU 103
MET 115
THR 145
CYS 189
CYS 188
Two important
The
subunits ofresidues
the nAChR
in the
binding
are
shown
pocket
with are
Nicotine
shown.
These the
inside
residues
binding
have
pocket
been
shown to interact with the
ligand.
Water in the Binding Pocket
Zone 2
Zone 1
 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
Zone 4
Zone 5
Zone 3
Water in the Binding Pocket
Water molecules which remain in
their position in the Binding Pocket
with Nicotine bound
Docking
 Docking various ligands such as
nicotine, acetylcholine,
imidacloprid (an insecticide) onto
AChBP and the α7 nAChR to look
at possible binding modes
Nicotine
docked onto
the AChBP
binding site
 An automated docking program
docks ligands onto hundreds of
frames from a trajectory
Nicotine
Carbamylcholine
HEPES
Results

Structure Generation:
Determined the structure of the α7 nAChR and several related proteins such as
GABAA, 5HT3, and Glycine receptor and used the models for various structural
studies

Molecular Dynamics:
i. Molecular dynamics studies of a homologue of the ligand binding (LB) domain of
nAChR with Nicotine, Carbamylcholine, and HEPES
ii. Studying the role of water in the binding pocket
iii. MD of α7 mutants

Coarse-graining:
i. GNM
ii. CONCOORD
iii. Grouping of eigenvectors from MD trajectories

Automated Docking:
Automated docking of ligands (Nicotine, acetylcholine, carbamylcholine, insecticides)
onto AChBP and nAChR (and its mutants) along a trajectory
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
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
Coarse-grain methods (3)
Grouping eigenvectors
 simplifying Molecular dynamics
data by grouping the eigenvectors
from the resulting trajectory.
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
TRP 144
LEU 103
MET 115
THR 145
CYS 189
CYS 188
The important residues in the
binding pocket are shown.
These residues have been
shown to interact with the
ligand.
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.