Watching the release of a photopharmacological drug from tubulin using time-resolved serial crystallography
Maximilian Wranik, Tobias Weinert, Chavdar Slavov, Tiziana Masini, Antonia Furrer, Natacha Gaillard, Dario Gioia, Marco Jacopo Ferrarotti, Daniel James, Hannah Glover, Melissa Carrillo, Demet Kekilli, Robin Stipp, Petr Skopintsev, Steffen Brünle, Tobias Mühlethaler, John H. Beale, Dardan Gashi, Karol Nass, D. Ozerov, Philip J. M. Johnson, Claudio Cirelli, Camila Bacellar, Markus Braun, Meitian Wang, Florian Dworkowski, Christopher J. Milne, Andrea Cavalli, Josef Wachtveitl, Michel O. Steinmetz, Jörg Standfuss
Abstract
The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we capture the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.