1. Molecular Dynamics Simulations and Docking Studies of AChBP and the Ligand Binding Domain of α7 nAChR Shiva Amiri JC 20-04-2005

2. 1.Simulation studies of AChBP with Nicotine, Carbamylcholine, and HEPES as ligands > also one simulation of the ligand binding domain of α7 nAChR 2.Docking studies of α7 nAChR with Nicotine, Imidacloprid (an insecticide), and Acetylcholine (ACh)

3. nAChR  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

4. 4Å structure of nAChR Ligand binding domain (LB)  core of 10 β-strands, forming a β- sandwich  an N-terminal α-helix, two short 3 10 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

5. AChBP  AChBP – from Lymnaea stagnalis, high homology with the ligand binding domain of ligand gated ion channels (LGICs) i.e. nAChR, GABA, Glycine, 5- HT 3 > Highest sequence identity with homomeric nAChR Celie et al., Neuron, March 2004 AChBP with HEPES (1UX2) – 2.1 Å AChBP with Carbamylcholine (1UV6) – 2.5 Å AChBP with Nicotine (1UW6) – 2.2 Å

6. List of simulations apo AChBP (1UX2)10 ns apo AChBP (1UW6)10 ns apo AChBP (1UV6)10 ns apo α7 nAChR LB domain (model) 10 ns AChBP (1UX2) with HEPES 10 ns AChBP (1UW6) with Nicotine 10 ns AChBP (1UV6) with Carbamylcholine 10 ns

7. The Ligands …  Nicotine  Carbamylcholine  HEPES ACh derivative, 10-fold less binding affinity for AChBP compared to ACh successful binding under crystallization conditions Very high affinity for both nAChR and AChBP

8. Making the topologies…  InsightII was used for protonating the ligands and Spartan was used to get the charges  Further details on making a topology on http://indigo1.biop.ox.ac.uk/wiki/index.php/Making_a_topology_file_- _a_quick_guidehttp://indigo1.biop.ox.ac.uk/wiki/index.php/Making_a_topology_file_- _a_quick_guide  For HEPES, I used PRODRG2.5 (beta), it gives GROMOS96 topologies > have to check the topologies produced by this server…there are some bugs  A 1 ns simulation in water was run on each ligand after making its topology before including it with the protein

9. apo AChBP (1UV6)  Crystal structure had two Carbamylcholines bound in binding site in two adjacent subunits  Higher covariance near TM domain, in subunits where the ligands were bound in crystal structure  GNM run showing highest flexbility of ligand binding region, as well as the bottom where the LB domain joins the TM domain http://s12-ap550.biop.ox.ac.uk:8078/dynamite_html/index.htm l

10. AChBP + nicotine  rmsf plot (rmsf values as B-factor values)  the region nearest the TM domain, and the ligand binding site are most flexible as well as the very top of the receptor

11. AChBP + Nicotine: PCA  Covariance line plot (70%) (top view)  Heavier covariance in two of the 5 subunits  Covariance line plot (80%) (side view)  Heavier covariance at the very top and the very bottom of subunits, where it meets the TM domain  porcupine plot of the first eigenvector (top view)  larger eigenvalues in two of the 5 subunits http://s12-ap550.biop.ox.ac.uk:8078/dynamite_html/index.htm l  agrees with simulations of AChBP bound to Ach where only 2 ACh molecules are required to keep AChBP in ligand bound state rather than 5 (Gao et al., J. Biol. Chem, 2005)

12. The Binding Site  Ligands bind in the interface between two subunits > the principal (+) side composed of loops A, B, C and the complementary side (-) composed of loops D and E  Ligand is completely buried in the protein Brejc et. al., Nature, May 2001 Ligand sitting behind the C-loop of the principal side of the receptor

13. Nicotine binding  Hydrophobic interactions with surrounding residues  Hydrogen bonding with Ser349, Trp350  it is thought that the bridging water molecules with Leu515 and Met527 contribute significantly to the binding of NCT

14.  Figure showing the hydrophobic interactions mostly exist between Trp350 and Nicotine  Also between cys395 and Nicotine

15.  ‘Breathing’ motion  Gain of symmetry upon ligand binding? First principal component Nicotine in binding pocket  Nicotine is stationery at its protonated N

16. Next …  Docking of ligands every x frames to look at binding behaviour throughout the simulation (using AUTODOCK) Nicotine docked onto the binding pocked of AChBP

17. Docking studies of α7 nAChR Some α7 background:  Homopentameric cationic channel  Found in central nervous system  Implicated in learning disabilities, Parkinson’s, Alzheimer’s, alcoholism, and nicotine addiction Docking:  The ligand binding domain is used for the docking studies with AUTODOCK  Modelled on new AChBP HEPES bound structure (2.1 Å) (Celie et al., Neuron, March 2004) using MODELLER  Nicotine (NCT), Acetylcholine (ACh), and Imidacloprid (IMI) used as ligands

18. Leu118  Leu118 is believed to be involved in the selectivity and binding of agonists  Docking carried out with wild type (WT), and L118D, L118E, L118K, L118R mutations for all three ligands

19. WT and Mutations for Nicotine  Binding energies for mutations (lowest to highest): E, D, R, K  all 50 WT docks in the exact same position in binding site L118R L118E L118D L118K

20. ACh WT Lowest energy dock  Smaller molecule, may be able to bind in different orientations  Simulation studies of ACh with α7 nAChR reveal very mobile behaviour of ACh in binding pocket (Henchman et al., Biophys. J., April 2005)

21. ACh and NCT binding  The lowest energy, highest ranked docks of NCT and ACh puts the ammomium group in the same position NCT boundACh bound NCT and ACh superimposed

22. Further docking  Fighting with Imidacloprid docks…  More ACh docking to look for a more clear pattern  Using the 4Å Torpedo marmorata (Unwin, Journal of Molecular Biology, March, 2005) structure for docks to compare binding sites and modes of ligand binding

23. Summary + Future Directions Simulations  Simulations show highest covariance and flexibility near the TM domain, in ligand binding site, and at the very top of the receptor  Higher covariance in subunits with bound ligand, even in APO simulations  First eigenvector shows ‘breathing motion’ in agreement with Henchman’s data  Further analysis on individual subunits, binding site, ligand contacts and behaviour needs to be done Docking  Mutations cause incorrect binding orientations of nicotine  ACh … multiple binding modes?  IMI in progress  Heteropentameric EM structure will be used for further docking and comparison of different binding sites

25. L118DL118E L118KL118R