The dynamic interplay between proteins and their ligands is central to molecular biology, pharmacology, and drug development but is difficult to resolve experimentally. Using time-resolved serial crystallography at a synchrotron and X-ray laser, we studied the release of the photochemical affinity switch azo-Combretastatin A4 from the anti-cancer target tubulin. Thirteen logarithmically spaced temporal snapshots at near-atomic resolution are complemented by time-resolved spectroscopy and molecular dynamics simulations. They show how the photoinduced cis to trans isomerization of the azobenzene bond stretches the ligand in the picosecond to nanosecond range, followed by stepwise opening of a gating loop within microseconds, and completion of the unbinding reaction within milliseconds. Ligand unbinding is accompanied by collapse of the binding pocket and global tubulin-backbone rearrangements. Our results have implications for the molecular basis of photopharmacology, the mechanism of action of anti-tubulin drugs and provide a general experimental framework to study protein-ligand interaction dynamics.

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