Molecular Dynamics of AChBP: Water in the Binding Pocket

Shiva Amirihttp://sbcb.bioch.ox.ac.uk/amiri.php

Biophysical Society Annual Meeting, February, 2006

AChBP: nAChR Ligand Binding Domain Homologue

Ligand binding domain (LB)

Transmembrane domain (TM)

Intracellular domain (IC)

Unwin, Journal of Molecular Biology, 2005

nAChR AChBP

Celie et. al, Neuron, 2004

Ligand binding pocket

• a ligand gated ion channel (LGIC) found in central and peripheral nervous system

• mutations lead to various diseases such as epilepsy, myasthenic syndromes, etc.

• implicated in Alzheimer’s disease and Parkinson’s disease

• mediates nicotine addiction

Loop A

Loop B

β1-β2 Loop

Loop G

Loop E

Loop F

Loop C

CYS Loop

Loop D

• Studying the behaviour of the binding pocket in the presence and absence of ligands

1. The structure of the binding pocket (distances, dihedrals, structural integrity)

2. The role of water in the binding of ligand to the binding site

LEU103

THR 145

TRP 144CYS 189

MET 115

CYS 188

These residues interact with the ligand either directly or via bridging waters

• Molecular Dynamics (MD) simulations of AChBP using GROMACS (GROMOS96)

• Focus on structural changes and ligand/protein interactions in the binding pocket

Molecular Dynamics

• 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

• 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)

MSF Block Analysis

0 2 4 6 8

Time (ns)

MS

F (

Å)

1UW6 without Nicotine

1UW6 with Nicotine

1UV6 without Carbamylcholine

1UV6 with Carbamylcholine

Global Motions0

.6

0.8

1

1

.2

1.4

Binding Pocket Motions

0 2 4 6 8 10

0

1

2

3

RM

SD

(Å

)

Binding pocket of 1UW6 with and without Nicotine bound

0

1

2

3

RM

SD

(Å

)

Time (ns)

Binding pocket of 1UV6 with and without Carbamylcholine bound

0 2 4 6 8 10Time (ns)

• Several atoms involved in the binding of the ligand were used to carry out RMSD calculations

• The residues of the binding pocket are more constrained in the presence of a ligand

without ligand

with ligand

• Higher density of water molecules in the binding sites of ligand bound AChBP

Time-averaged water density plots for AChBP with Carbamylcholine bound

Persistent Waters

Binding pockets

Persistent Waters

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

• Several zones identified in the binding site where water molecules persist for >= 40 % of the duration of the simulation

Water densities in the binding site

Zone 1Zone 2

Zone 4

Zone 3

Zone 5

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

Time (ns)

Time (ns)

Dis

tanc

e (

Å)

Distance between LEU103 and MET115 on loop E

Dis

tanc

e (

Å)

Waters Between LEU103 and MET115

Bridging Waters

• Following waters in one of the zones of persistent waters.

- A water situated between LEU103 and MET115 leaves the site and is instantly replaced by another water molecule

- When both waters are gone, the space between the two residues is decreased and the interactions with the ligand are affected (decreased)

Conclusions

• AChBP has greater global flexibility in the non-ligand bound state

- the binding of a ligand adds structural integrity to the ion channel

• The binding pocket is less flexible in the presence of a ligand

• There are positionally conserved waters in the binding pocket, higher in quantity and more persistent in the presence of a ligand

• Several water molecules bridge the ligand to neighbouring residues in the binding site

• These waters plays a structural role in the binding pocket, adding rigidity that may extend beyond the binding site to functionally relevant loops

Acknowledgements• Prof. Mark S. P. Sansom• Dr. Philip C. Biggin

• 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

• 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

Fre

qu

en

cy

(H

z)

-200 -100 0 100 200

Fre

qu

en

cy

(H

z)

-200 -100 0 100 200

The Ligands

NH

+

N CH3

Nicotine

• Nicotine is less flexible in the binding pocket than carbamylcholine

• There seems to be one mode of binding for Nicotine

Carbamylcholine

O

ON

+CH3

H2NCH3

CH3

Distance between CYS 188 and THR 145

Carbamylcholine

Subunit 1

Subunit 2Subunit 3

Subunit 4Subunit 5

NicotineDistance between CYS 188 and THR 145

Distance between CYS 188 and THR 145

APO NicotineDistance between CYS 188 and THR 145

APO Carbamylcholine

Dis

tan

ce (Å

)

Time (ns)

Distances between residues in the BP

Loop C of AChBP (1UV6) With and Without Carbamylcholine

Water molecule Residue 1 Residue 2

Number of occurences

SOL1256 MET 527 NCT 18814 1870

SOL7253 MET 527 NCT 18814 1342

SOL1078 MET 115 NCT 18817 1056

SOL1078* TRP 968 NCT 18817 620

SOL5732* NCT 18813 SER 143 308

SOL13378 NCT 18817 TRP 968 188

SOL1256* TRP 350 NCT 18814 147

SOL7253* TRP 350 NCT 18814 130

SOL17829 TYR 165 NCT 18817 100

SOL6834 NCT 18816 TRP 762 79

Water molecule Residue 1 Residue 2

Number of Occurences

SOL9297 MET 729 CCE 1026 2003

SOL14171* CCE 1027 TRP 758 1387

SOL14171 CCE 1027 TYR 807 1386

SOL1428 CCE 1026 TYR 602 580

SOL6702 CCE 1027 TRP 758 505

SOL1428** CCE 1026 TRP 553 429

SOL1428* CCE 1026 THR 554 403

SOL6702* CCE 1027 TYR 807 377

SOL11808 MET 934 CCE 1027 354

SOL16192* CCE 1027 TYR 807 315

Nicotine Carbamylcholine