1. 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

2. AChBP: nAChR Ligand Binding Domain Homologue Ligand binding domain (LB) Transmembrane domain (TM) Intracellular domain (IC) Unwin, Journal of Molecular Biology, 2005 nAChRAChBP 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

3. 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 144 CYS 189 MET 115 CYS 188 These residues interact with the ligand either directly or via bridging waters

4. 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 SimulationPDB codeLigand? NCT1UW6Nicotine NCT-Apo1UW6- CCE1UV6Carbamylcholine CCE-Apo1UV6- * All simulations were run for 10 ns

5. 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) MSF (Å) 1UW6 without Nicotine 1UW6 with Nicotine 1UV6 without Carbamylcholine 1UV6 with Carbamylcholine Global Motions 0.6 0.8 1 1.2 1.4

6. Binding Pocket Motions 0 2 4 6 8 10 0 1 2 3 RMSD (Å) Binding pocket of 1UW6 with and without Nicotine bound 0 1 2 3 RMSD (Å) Time (ns) Binding pocket of 1UV6 with and without Carbamylcholine bound 0 2 4 6 8 10 Time (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

7. 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

8. Persistent Waters ZONEAverage % for NCT Average % for CCE 19292.5 24579.5 34089.5 46076 55550 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 1 Zone 2 Zone 4 Zone 3 Zone 5

9. Water molecules which remain in their position in the binding pocket with Nicotine bound

11. 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

12. Time (ns) Distance between LEU103 and MET115 on loop E 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)

13. 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

14. 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

15. Frequency (Hz) -200 -100 0 100 200 Frequency (Hz) -200 -100 0 100 200 The Ligands Nicotine Nicotine is less flexible in the binding pocket than carbamylcholine There seems to be one mode of binding for Nicotine Carbamylcholine

16. Distance between CYS 188 and THR 145 Carbamylcholine Subunit 1 Subunit 2 Subunit 3 Subunit 4 Subunit 5 Nicotine Distance between CYS 188 and THR 145 APO Nicotine Distance between CYS 188 and THR 145 APO Carbamylcholine Time (ns) Distances between residues in the BP

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

18. Water moleculeResidue 1Residue 2 Number of occurences SOL1256MET 527NCT 188141870 SOL7253MET 527NCT 188141342 SOL1078MET 115NCT 188171056 SOL1078*TRP 968NCT 18817620 SOL5732*NCT 18813SER 143308 SOL13378NCT 18817TRP 968188 SOL1256*TRP 350NCT 18814147 SOL7253*TRP 350NCT 18814130 SOL17829TYR 165NCT 18817100 SOL6834NCT 18816TRP 76279 Water moleculeResidue 1Residue 2 Number of Occurences SOL9297MET 729CCE 10262003 SOL14171*CCE 1027TRP 7581387 SOL14171CCE 1027TYR 8071386 SOL1428CCE 1026TYR 602580 SOL6702CCE 1027TRP 758505 SOL1428**CCE 1026TRP 553429 SOL1428*CCE 1026THR 554403 SOL6702*CCE 1027TYR 807377 SOL11808MET 934CCE 1027354 SOL16192*CCE 1027TYR 807315 Nicotine Carbamylcholine