1. Bioinformatics and Molecular Modeling studies of Membrane Proteins Shiva Amiri

2. Problem: Difficult to obtain high resolution crystallographic images of membrane proteins Structure Determination Unwin et.al, Nature, 26 June 2003

3. Getting there?  Some success using cryo-electron microscopy coupled with Fourier Transforms, i.e. Unwin’s 4 Å image of the TM region.  But still no full structure Unwin et al., Nature, 26 June 2003

4. My Project  To design structure determination software for Ligand Gated Ion Channels (LGICs) i.e. nAChR, GABA A and GABA C receptors, 5HT 3 receptor, Glycine receptor

5. Main focus so far: The α-7 Nicotinic Acetylcholine Receptor (nAChR)  cationic channel  homopentamer  four transmembrane regions (M1-M4)

6. Transmembrane Domain Alignment Homology modeling – Transmembrane domain

7.  The homology model of the TM region with the Torpedo Marmorata structure (PDB: 1OED - 4 Å) and the Chick α-7 sequence using MODELLER

8. Ligand Binding Domain Alignment Homology Modeling – Ligand Binding domain

9.  The homology model of the LBD with Acetylcholine Binding Protein (AChBP) as the structure (PDB: 1I9B – 2.7 Å) and the Chick α-7 sequence using MODELLER

10. The software …  Combining the Transmembrane domain with the Ligand Binding domain  Failed first attempt: Minimizing distances between target residues in the LBD and the TM domains - 6 degrees of freedom (rotations and translations on all three axes) - Models were not straight

11. Second attempt … New algorithm : z-axis b.Align each domain onto the z-axis a.straighten each domain with respect to the z-axis

12. c.Rotate and translate about z-axis - angle of rotation and steps of translations are user- defined z-axis Theta (angle of rotation) Second Attempt continued …

13.  At each rotation and for each translation the Unwin distance, the Termini distance the number of bad contacts is calculated

14. Scoring Functions 1.Unwin Distance – the distance between residues from the TM domain and the LB domain that are meant to come into close proximity

15. 2.Termini Distance – the distance between the C- terminus of the LB domain and the N-terminus of the TM domain Scoring Functions continued …

16. 3.Bad Contacts – Number of residues that are closer than a certain cut-off distance (user-defined), currently set to 5 Å Scoring Functions continued…

17. Plots of Scoring Functions Unwin DistanceTermini Distance Bad contacts

18. Linear Combinations of Scoring Functions Unwin + TerminiUnwin + Termini + Bad contacts

19. Choosing the Best Model  Model chosen based on scoring function data  Once a good model was decided on, energy minimization using GROMACS was carried out to ensure the electrochemical legitimacy of the model

20. Model of the α-7 nAChR

21. Gaussian Network Model (GNM) Analysis  A course-grained model to approximate molecular motions of proteins  Current code cannot allocate memory for the 1660 residues of the α-7 nAChR  Analysis has been done using the TM domain and the LB domain separately  GNM was also run on one subunit of the model  B-values generally in agreement with crystallographic data but modeled structures are difficult to analyze using present code

22. AChBP – one subunit

23. α-7 nAChR Transmembrane

24. α-7 nAChR model – 2 subunits

25. CONCOORD Analysis  Generates protein conformations around a given structure based on distance restrictions  Principal Component Analysis (PCA) is applied on the 500 resulting structures from CONCOORD  First eigenvector shows opening and closing of the pore as the subunits rotate

26. First eigenvector

27. Eigenvector PlotCovariance lines

28. Future work…  Looking at the hydrophobic girdle (M2) of LGICs to study patterns of conservation and the behaviour of these residues during gating  Further verification and analysis of models  Other models of LGICs Summary  Software designed to determine structure of LGICs  Structure of α-7 nAChR  Used various methods (GNM, CONCOORD) to look at possible motions using the hypothesized structure

29. Thanks to: Prof. Mark S.P. SansomSundeep Deol Dr. Phil BigginYalini Pathy Dr. Kaihsu Tai Jonathan Cuthbertson Dr. Paul Barrett Pete Bond Jeff Campbell Dr. Alessandro GrotessiKatherine Cox Dr. Daniele BemporadJennifer Johnston Dr. Jorge PikunicRobert D’Rozario Dr. Shozeb HaiderLoredana Vaccaro Dr. Andy HungJohn Holyoake Dr. Bing WuTony Ivetac Oliver BecksteinSylvanna Ho Syma KhalidSamantha Kaye Zara Sands George Patargias

31. Hydrophobic girdle M2 alignment

32. b.Align each domain onto the z-axis

33. c.Rotate and translate about z-axis - angle of rotation and steps of translations are user- defined

35. α-7 nAChR LBD – 3 subunits