Discovery and Characterization of the Laulimalide-Microtubule Binding Mode by Mass Shift Perturbation Mapping

Conventional approaches to site mapping have so far failed to identify the laulimalide binding site on microtubules. Using mass shift perturbation analysis and data-directed docking, we demonstrate that laulimalide binds to the exterior of the microtubule on β-tubulin, in a region previously unknown to support ligand binding and well removed from the paclitaxel site. Shift maps for docetaxel and laulimalide are otherwise identical, indicating a common state of microtubule stability induced by occupancy of the distinct sites. The preferred binding mode highlights the penetration of the laulimalide side chain into a deep, narrow cavity through a unique conformation not strongly populated in solution, akin to a “striking cobra.” This mode supports the development of a pharmacophore model and reveals the importance of the C1–C15 axis in the macrocycle. ► The laulimalide binding site exists on the exterior of β-tubulin in microtubules ► The laulimalide binding mode is characterized to support further analog development ► Combining mass shift perturbation and data-directed docking represents a powerful method for protein-ligand mapping