1. Towards a Molecular Description of the GABA-A and Glycine Neuroreceptors. Jeanette Hobbs

2. Overview Introduction –Justification –What are GABA & Glycine? –Neurotransmitters –Neuroreceptors –Ligand-gated Ion Channels –The GABA-A & glycine receptor –Benzodiazepine binding site Experimental - Crystallization –Objective –Previous attempts –Protein Purity –Crystallization Screens

3. No structural data YET, targets of pharmacologically and clinically important drugs, e.g. benzodiazepines (BZ). WHY? Bottleneck in identification of protein elements that constitute pharmocophore sites, hampering structure- based drug design.

4. Introduction-neurotransmitters Chemically four classes of neurotransmitters: –Acetylcholine –Excitatory amino acids – glutamate and Inhibitory – GABA & glycine –Amines – seratonin, dopamine, histamine –Neuropeptides - endorphins GABA is: gamma-amino-butyric acid

6. Glycine The brain stem and spinal cord

7. What do Neurotransmitters do? Neurotransmitters ferry information from the end of one nerve to the "beginning" of another by activating a large molecule at the far end of the synapse called a neuroreceptor. Direction of Impulse

9. Receptor is the lock – GABA-A or glycine Neuroreceptors Transmitter is the key – GABA or glycine A transmitter that binds briefly to a receptor, causing channels to open and ions to move in or out of that neuron = ligand-gated ion channel neuroreceptors

10. Neuroreceptors contd. Activation causes a net change in the electrical properties (membrane potential) of that neuron and determines its activity. Increase in Chloride ions = HYPERPOLARIZATION Neurons less likely to fire. Calming, tranquilizing, prevents us being overwhelmed by stressful situations!

11. Ligand-gated ion channel neuroreceptors Cell membrane neurotransmitter NT Channel closed Cl -

12. Ligand-gated ion channel neuroreceptors NT pore Cl -

13. Ligand-gated ion channel neuroreceptors NT

14. Ligand-gated ion channel neuroreceptors NT

15. The GABA-A Receptor ? Major mammalian inhibitory neurotransmitter receptor Pentameric integral membrane protein containing an ion channel selective for chloride ions. Extracellular part Transmembrane part

17. Target of numerous clinically used drugs, e.g. the benzodiazepines. Sedative, anxiolytic, anticonvulsive and myorelaxant effects

19. Benzodiazepine Binding Site Allosterically stimulate the function of the GABA-A receptor.

20. Allosteric stimulation Binds at its own separate site away from the active site.

21. Allosteric stimulation NT Valium Cl- NT Cl-

22. Benzodiazepine receptor-ligand interactions

23. GABA-A and Benzodiazepine Structure of drug binding sites and differences in different receptor isoforms are NOT KNOWN, as is the overall receptor structure. Do know about a homologous protein complex, the nicotinic acetylcholine receptor (does not recognize benzodiazepine). However, has been studied extensively. Structure has been determined based on: –Negatively stained 2-dimensional crystals –Electron microscope image analysis –To 4.6 Å

24. Nicotinic acetylcholine receptor at 4.6Å resolution: transverse tunnels in the channel wall. Miyazawa, A., Fujiyoshi, Y., Stowell, M. & Unwin, N. J. Mol. Biol. 288, 765- 786. (1999).

25. Three-dimensional view obtained from the fully averaged structure

26. Cross-sections through the fully averaged structure running along the central axis of the receptor, and rotated about this axis to bring in successive 5-fold related views

27. Objectives for overall project Find structure of different conformational states of the binding pocket for the drugs of the benzodiazepine types. To determine the differences of this pocket in different receptor subtypes. Express the receptor subunit fragments in E.coli followed by crystallization and 3-dimensional crystallographic analysis.

28. How? Dr. H. Xue Domain recognition, expression and purification Dr. M. Goeldner Synthetic Organic Chemistry Dr. E. Sigel Mutagenesis, expression, ligand binding studies Dr. J. Hobbs Crystallization of GABA-A receptor residue. Prof. P. Kuhn Crystallography

29. Experimental Details Structural elucidation impeded by large size of receptor. Need a minimal domain as the first step in dissecting the receptor structure. 131-residue fragment in the extra cellular region - Cys166 to Leu296 of  1 subunit of bovine GABA-A receptor. Contains five residues shown to be associated with BZ modulation of receptor function. Full-length polypeptide of 456 amino acid residues, the 131- residue is identical in sequence to the corresponding region of the human homolog.

30. Heteromers of the GABA-A receptor

31. Larger pore Smaller pore pentamer hexamer tetramer

32. Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis SDS PAGE of GABA-A and Glycine Sample

33. Laser Scattering for GABA-A protein residue

34. For Glycine Protein residue

35. Crystallization Screens Crystallization screens from Emerald Biostructures-Wizard I & II, and Cryo I & II. Crystal Growth Matrices are sets of 48 unique solutions for macromolecular crystal growth.

36. Wizard I & II Highly effective random sparse matrices Sixteen different crystallants. Eleven different buffers, ranging from pH 4.2 to10.5, ensure a broad sampling of crystallization space.

37. Cryo I & II Every formulation will flash-freeze to a clear amorphous glass in liquid nitrogen or in the cryo-stream at 100K. Eleven different cryocrystallants and sparing use of glycerol ensures a broad sampling of possible cryo conditions. Crystals can be frozen directly from their growth chambers, avoiding the additional step of pre-equilibration with an artificial cryo-solvent that can damage the crystal.

38. Questions ? Electrophoresis kit to test for purity of protein once it has been delivered here? Is the protein we have still OK to use > 2 mths old @ 4°C? Do we have an endless supply of the protein?