Theoretical binding studies of the clostridial tetanus toxin and the ganglioside GD1b

The goal of this project was to clarify the mechanism of action of Clostridium tetani toxin through the study of its binding processes with the native ligand, the membrane-bound neuronal ganglioside GD1b, using computational methods such as homology modeling, molecular dynamics simulations, and rigid body ligand docking. This study is expected to contribute to the identification of molecular targets for disease control through the study of the carbohydrate components of the ganglioside ligand as well as the development of schemes for docking carbohydrates to proteins. Tetanus is a member of the general A/B class of toxins with separate ligand binding and catalytic domains. Initial binding proceeds through a multistep transitional pathway, enabling a first low affinity contact with a membrane-bound ganglioside and a second high affinity contact with a membrane-bound receptor. In toxins of the Clostridium family, the toxin remains outside the cell until the binding event induces endocytosis. Characterization of the dynamics of this process is difficult because of the essential role of the membrane. Static methods such as x-ray crystallography can provide insights into initial and final states, but the size and complexity of the protein prohibits structural and dynamic studies by physical methods such as nuclear magnetic resonance spectroscopy. However, computational methods have proven a viable approach for the study of binding and the accompanying dihedral transitions that are important in conformational research. This dissertation focuses on the initial stages of the process, i.e., the first ganglioside binding/release step to determine ligand binding positions and putative dihedral transitions and equilibrium distributions of low energy conformers of the components of the ganglioside GD1b. This project necessitates the testing and modification of tools for computational methods, including the requisite development of a novel force field parameter set for carbohydrates in the CHARMM (Chemistry at Harvard Molecular Mechanics) computational package and analytical docking methods with validation against known carbohydrate/protein complexes. Overall, the project proceeds on three levels: identification of the carbohydrate binding domain on the tetanus toxin; building, parameterization, and molecular dynamics simulations of the components of the ganglioside ligand; and examination of the binding process through original docking schemes.