Transcript Molecular Dynamics of AChBP: Water in the Binding Pocket

Molecular Dynamics of AChBP: Water in the
Binding Pocket
Shiva Amiri
http://sbcb.bioch.ox.ac.uk/amiri.php
Biophysical Society Annual Meeting,
February, 2006
AChBP: nAChR Ligand Binding Domain Homologue
nAChR
AChBP
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Ligand binding domain (LB)
a ligand gated ion channel (LGIC)
found in central and peripheral
Ligand
nervous system
binding
pocket
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mutations lead to various diseases
such as epilepsy, myasthenic
syndromes, etc.
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implicated in Alzheimer’s disease and
Parkinson’s disease
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mediates nicotine addiction
Transmembrane domain
(TM)
Intracellular domain (IC)
Unwin, Journal of Molecular
Biology, 2005
Celie et. al, Neuron, 2004
Loop A
• Studying theLEU103
behaviour of the
binding pocket in the presence
and absence
of ligands
THR 145
Loop B
MET 115
Loop F
Loop C
Loop E
Loop D
2. The role of water in the binding of
ligand to the binding site
Loop G
CYS Loop
1. The structure of the binding
pocket (distances, dihedrals,
CYS 188
TRP 144
structural integrity)
CYS 189
β1-β2 Loop
These residues interact with the ligand
either directly or via bridging waters
Molecular Dynamics
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Molecular Dynamics (MD) simulations of
AChBP using GROMACS (GROMOS96)
Focus on structural changes and
ligand/protein interactions in the binding
pocket
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Describe the forces on all atoms:
•bonded (bonds, angles, dihedrals)
•non-bonded (van der Waals,
electrostatics)
• Result: positions of all atoms during a few
nanoseconds
Simulation
PDB code
Ligand?
NCT
1UW6
Nicotine
NCT-Apo
1UW6
-
CCE
1UV6
Carbamylcholine
CCE-Apo
1UV6
-
* All simulations were run for 10 ns
Global Motions
1.4
MSF Block Analysis
1.2
1UW6 without Nicotine
1UV6 without Carbamylcholine
1
0.8
1UV6 with Carbamylcholine
0.6
MSF (Å)
1UW6 with Nicotine
0
2
4
6
8
Time (ns)
• Simulations with ligands have lower mean square fluctuation (MSF)
values than those without ligand
• 10 ns is not enough to see the full range of motions involved in the
function of the receptor (ie. channel gating)
Binding Pocket Motions
• Several atoms involved in the binding of the ligand were used to carry
out RMSD calculations
Binding pocket of 1UW6 with
and without Nicotine bound
3
1
2
RMSD (Å)
2
1
0
0
RMSD (Å)
3
Binding pocket of 1UV6 with and
without Carbamylcholine bound
0
2
4
6
Time (ns)
8
10
• The residues of the binding
pocket are more constrained in the
presence of a ligand
0
2
4
6
Time (ns)
without ligand
with ligand
8
10
Persistent Waters
Time-averaged water density plots for AChBP with Carbamylcholine bound
Binding pockets
• Higher density of water molecules in the binding sites of ligand
bound AChBP
Persistent Waters
• Several zones identified in the
binding site where water molecules
persist for >= 40 % of the duration of
the simulation
Zone 2
Zone 1
Zone 4
Zone 5
Zone 3
ZONE
Average %
for NCT
Average %
for CCE
1
92
92.5
2
45
79.5
3
40
89.5
4
60
76
5
55
50
Water densities in the binding site
Water molecules which remain in
their position in the binding pocket
with Nicotine bound
Bridging Waters
• Ligand-protein interactions via
water molecules in the binding site
• Many of these waters remain for
>=40% (some > 90%) of the
simulation, suggesting functional
importance
Bridging Waters
Waters Between LEU103 and MET115
• Following waters in one of the zones
of persistent waters.
Distance (Å)
- A water situated between LEU103 and
MET115 leaves the site and is instantly
replaced by another water molecule
Time (ns)
Distance (Å)
Distance between LEU103 and MET115 on loop E
Time (ns)
- When both waters are gone, the space
between the two residues is decreased and
the interactions with the ligand are affected
(decreased)
Conclusions
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AChBP has greater global flexibility in the non-ligand bound state
- the binding of a ligand adds structural integrity to the ion channel
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The binding pocket is less flexible in the presence of a ligand
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There are positionally conserved waters in the binding pocket, higher in quantity and
more persistent in the presence of a ligand
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Several water molecules bridge the ligand to neighbouring residues in the binding site
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These waters plays a structural role in the binding pocket, adding rigidity that may
extend beyond the binding site to functionally relevant loops
Acknowledgements
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Prof. Mark S. P. Sansom
Dr. Philip C. Biggin
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Dr. Alessandro Grottesi
Dr. Kaihsu Tai
Dr. Zara Sands
Dr. Oliver Beckstein
Dr. Jorge Pikunic
Dr. Andy Hung
Dr. Shozeb Haider
Dr. Syma Khalid
Dr. Pete Bond
Dr. Kia Balali-Mood
Dr. Hiunji Kim
Dr. Martin Ulmschneider
Dr. Daniele Bemporad
Dr. Bing Wu
Sundeep Deol
Yalini Pathy
Jonathan Cuthbertson
former members
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Jennifer Johnston
Katherine Cox
Robert D’Rozario
Jeff Campbell
Loredana Vaccaro
John Holyoake
Tony Ivetac
Samantha Kaye
Sylvanna Ho
Benjamin Hall
Tim Carpenter
Emi Psachoulia
Chze Ling Wee
Ranjit Vijayan
Michael Kohl
The Ligands
N
+
+
N
H
H2N
N
O
CH3
CH3
Carbamylcholine
Frequency (Hz)
Frequency (Hz)
Nicotine
-200
CH3
O
CH3
-100
0
100
200
-200
-100
0
100
• Nicotine is less flexible in the binding pocket than carbamylcholine
• There seems to be one mode of binding for Nicotine
200
Distances between residues in the BP
Nicotine
Carbamylcholine
Distance between CYS 188 and THR 145
Distance between CYS 188 and THR 145
Distance (Å)
Subunit 1
Subunit 2
Subunit 3
Subunit 4
Subunit 5
APO Nicotine
APO Carbamylcholine
Distance between CYS 188 and THR 145
Distance between CYS 188 and THR 145
Time (ns)
Loop C of AChBP (1UV6) With and Without
Carbamylcholine
Carbamylcholine
Nicotine
Water
molecule
Residue 1
Residue 2
Number of
occurences
Water
molecule
Residue 1
Residue 2
SOL1256
MET
527
NCT 18814
1870
SOL9297
MET
CCE 1026
2003
SOL7253
MET
527
NCT 18814
1342
SOL14171*
CCE 1027
TRP
758
1387
SOL1078
MET
115
NCT 18817
1056
SOL14171
CCE 1027
TYR
807
1386
SOL1078*
TRP
968
NCT 18817
620
SOL1428
CCE 1026
TYR
602
580
SOL5732*
NCT 18813
SER
143
308
SOL6702
CCE 1027
TRP
758
505
SOL13378
NCT 18817
TRP
968
188
SOL1428**
CCE 1026
TRP
553
429
SOL1256*
TRP
350
NCT 18814
147
SOL1428*
CCE 1026
THR
554
403
SOL7253*
TRP
350
NCT 18814
130
SOL6702*
CCE 1027
TYR
807
377
SOL17829
TYR
165
NCT 18817
100
SOL11808
MET
CCE 1027
354
SOL6834
NCT 18816
TRP
79
SOL16192*
CCE 1027
TYR
315
762
729
934
807
Number of
Occurences