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Transcript
Towards a Molecular Description of the GABA-A and Glycine Neuroreceptors. Jeanette Hobbs 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 WHY? • No structural data • YET, targets of pharmacologically and clinically important drugs, e.g. benzodiazepines (BZ). • Bottleneck in identification of protein elements that constitute pharmocophore sites, hampering structurebased drug design. 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 • Glycine The brain stem and spinal cord 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 Neuroreceptors • Transmitter is the key – GABA or glycine • Receptor is the lock – GABA-A 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 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! Ligand-gated ion channel neuroreceptors Channel closed ClClCl- Cl- ClNT neurotransmitter ClCell membrane Cl- Ligand-gated ion channel neuroreceptors Cl- ClCl- pore Cl- Cl- Cl- Cl- ClCl- ClNT ClCl- Cl- ClCl- Cl- Cl- Cl- Cl- Ligand-gated ion channel neuroreceptors NT Ligand-gated ion channel neuroreceptors 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 • Target of numerous clinically used drugs, e.g. the benzodiazepines. • Sedative, anxiolytic, anticonvulsive and myorelaxant effects Benzodiazepine Binding Site • Allosterically stimulate the function of the GABA-A receptor. Allosteric stimulation • Binds at its own separate site away from the active site. Allosteric stimulation NT Cl- ClClCl- ClValium Cl- ClCl- NT ClClCl- Cl- Cl- Benzodiazepine receptor-ligand interactions 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 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, 765786. (1999). Three-dimensional view obtained from the fully averaged structure 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 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. How? Dr. M. Goeldner Synthetic Organic Chemistry Dr. J. Hobbs Crystallization of GABA-A receptor residue. Prof. P. Kuhn Crystallography Dr. E. Sigel Mutagenesis, expression, ligand binding studies Dr. H. Xue Domain recognition, expression and purification 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 131residue is identical in sequence to the corresponding region of the human homolog. Heteromers of the GABA-A receptor Larger pore pentamer hexamer Smaller pore tetramer SDS PAGE of GABA-A and Glycine Sample • Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis Laser Scattering for GABA-A protein residue For Glycine Protein residue 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. 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. 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. 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?