Taccalonolide AJ uncouples tubulin GTP hydrolysis from microtubule depolymerization.

Microtubules are dynamic polymers of tubulin subunits that are vital for a variety of eukaryotic cell functions, including intracellular trafficking and cell division. Microtubule-targeting drugs are among the oldest and most widely used chemotherapeutics. Despite their extensive use, the development and optimization of these drugs is hindered by our limited understanding of the molecular impacts on microtubules. Recent high-resolution structures have begun to explain how paclitaxel (Taxol) stabilizes microtubules: it reverses the hydrolysis-driven lateral strain at the microtubule seam and induces an expanded, GTP-like conformational state that promotes polymerization. In this work, we demonstrate that the microtubule-targeting natural product taccalonolide AJ (taccaAJ) stabilizes microtubules in a manner completely distinct from paclitaxel. Using cryo-EM, we show that taccaAJ stabilizes a compacted, post-GTP hydrolysis state, which is usually prone to depolymerization. In vitro assays show that taccaAJ-stabilized microtubules are more rigid than paclitaxel-stabilized microtubules, and reconstituted dynamics with taccaAJ exhibit increased rescue close to the plus-end. Finally, we use fluorescent biosensors for the compacted tubulin conformation to confirm that taccaAJ-stabilized microtubules in cells are compacted yet stable against depolymerization. We propose that taccaAJ preferentially binds a GDP lattice, promotes lateral interactions between protofilaments, and thus creates a rigid, stable microtubule in a compacted, post-GTP hydrolysis state.