Computational studies of ligand-protein interactions. Part I: The T-Taxol conformation. Part II: Elucidating interdependent binding sites on tubulin

Part I: T-Taxol is a proposal for the bioactive conformation of paclitaxel (PTX) derived from fitting ligand conformations to the electron crystallography (EC) density of the tubulin-ligand complex. It has been confirmed by independent refinement of the PTX-tubulin structure, the activity of bridged T-Taxol analogs, and mutation studies in the yeast tubulin framework. Nonetheless, some structural ambiguities remain based on an uncritical interpretation of the solid state REDOR measurement of two internuclear 13C--19F distances in a fluorinated derivative of PTX. The issues are evaluated by an analysis of the static and dynamic properties of PTX and the PTX-tubulin complex, torsional force field parameters, and the error assigned to the REDOR distances (+/- 0.5 A). In addition, a proposed alternative to T-Taxol (PTX-NY) is shown to be incompatible with both the EC density and the activity of a highly active series of bridged taxanes. The development of series of bridged and simplified taxanes using the T-Taxol conformation resulted in analogs of the former that induce microtubule formation more efficiently. NAMFIS analysis, along with molecular dynamics simulations and docking, are used to analyze the compounds, and we propose that the enhanced ability to assemble microtubules by these taxane derivatives is linked to their ability to effectively shape the conformation of the M-loop of tubulin for cross-protofilament interaction.;Part II: A model of colchicine (COL) and 2-methoxyestardiol (2ME2) binding to tubulin was developed using molecular dynamics and docking. Available experimental data, including a number of labeling studies, supports the two-site hypothesis in which conformational changes in the H7--H8 loop open opposing sites with differing binding properties and affinities. The beta-Val236Ile mutation that causes resistance to 2ME2 with no cross-resistance to COL is located in the site designated for the former ligand and not near the site for the later one.