Building a Homology Model of the Transmembrane Domain of the Human Glycine α-1 Receptor Presented by Stephanie Lee Research Mentor: Dr. Rob Coalson Glycine Alpha 1 Receptor (GlyRa1) Member of the superfamily of ligand-gated ion channels Essential to the transfer of information within the nervous system Very sensitive to anesthetics and alcohol Anesthetics trigger alterations in transmembrane domain Structural problem Does not crystallize well (transmembrane) 1
Nicotinic Acetylcholine Receptor (nachr) Same pentameric superfamily Facilitates fast transmission of electrical signals Found in high concentrations at the nerve-muscle synapse Known 4Å structure nachr Structure Assembled from a ring of five homologous subunits Two binding sites Extracellular, transmembrane, intracellular domains Subunit-subunit interfaces provide potential pathways for diffusing ions 2
Transmembrane Domain Four α-helical segments M1-M4 Pore shaped by inner ring of M2 helices Outer shell of helices (M1, M3, M4) shield the inner ring from lipids nachr Gating Acetylcholine binds at both sites Local disturbance causes α-subunits to rotate Unlocks interactions that restrict the rotational movements of the pore-lining helices Destabilizes weak hydrophobic interactions holding the gate together Lumen widens causing the pore to open 3
Sequence Alignment Search protein databases to find which protein to model after Align sequences of nachr δ chain and GlyRa1 using ClustalW Model Membranes For each transmembrane, use Modeller to create 10 models Choose the models with the lowest Modeller Objective Function Use VMD Multiple Sequence Alignment to align each model with the corresponding segment of nachr δ chain Manually merge the separate pdb files 4
Fill Gaps Use Modeller to create 10 models different alignment file Choose model with lowest Modeller Objective Function Analyze Results Evaluate Discrete Optimized Potential Energy (DOPE) scores 5
Refine Model Refine loop using Modeller Choose model with lowest Modeller Objective Function as best model Analyze Results Compare DOPE scores for each residue of the two models 6
Analyze Results Plot Statistics 73 residues in most favored regions (93.6%) 4 residues in additional allowed regions (5.1%) 1 residue in generally allowed regions (1.3%) 0 residues in disallowed regions (0.0%) Direct Modeling Align complete sequences of nachr α δ subunit and GlyRa1 using ClustalW Use Modeller to generate 10 models transmembrane domain of the subunit Choose model with lowest Modeller Objective Function Duplicate subunit four times and rotate each copy 72 to create pentameric structure 7
Final Model Analyze Results Plot Statistics 405 residues in most favored regions (96.4%) 15 residues in additional allowed regions (3.6%) 0 residues in generally allowed regions (0.0%) 0 residues in disallowed regions (0.0%) 8
References Bertaccini, E. J., Shapiro, J., Brutlag, D. L., and Trudell, J. R. (2005) Homology Modeling of a Human Glycine Alpha 1 Receptor Reveals a Plausible Anesthetic Binding Site, J. Chem. Inf. Model. 45, 128-135. Law, R. J., Henchman, R. H., and McCammon, J. A. (2005) A gating mechanism proposed from a simulation of a human α7 nicotinic acetylcholine receptor, PNAS 102, 6813-6818. Ma, D., Liu, Z., Li, L., Tang, P., and Xu, Y. (2005) Structure and Dynamics of the Second and Third Transmembrane Domains of Human Glycine Receptor, Biochemistry 44, 8790-8800. Schofield, C. M., Trudell, J. R., and Harrison, N. L. (2004) Alanine- Scanning Mutagenesis in the Signature Disulfide Loop of the Glycine Receptor α1 Subunit: Critical Resides for Activation and Modulation, Biochemistry 43, 10058-10063. Unwin, N. (2005) Refined Structure of the Nicotinic Acetylcholine Receptor at 4Å Resolution, J. Mol. Biol. 346, 967-989. Acknowledgements Dr. Rob Coalson Dr. Mary Cheng Dr. Rajan Munshi Bioinformatics and Bioengineering Summer Institute NIH and NSF 9