Large-Scale Ligand Perturbations of the Protein Conformational Landscape Reveal State-Specific Interaction Hotspots

[Image: see text] Protein flexibility is important for ligand binding but often ignored in drug design. Considering proteins as ensembles rather than static snapshots creates opportunities to target dynamic proteins that lack FDA-approved drugs, such as the human chaperone, heat shock protein 90 (Hsp90). Hsp90α accommodates ligands with a dynamic lid domain, yet no comprehensive analysis relating lid conformations to ligand properties is available. To date, ∼300 ligand-bound Hsp90α crystal structures are deposited in the Protein Data Bank, which enables us to consider ligand binding as a perturbation of the protein conformational landscape. By estimating binding site volumes, we classified structures into distinct major and minor lid conformations. Supported by retrospective docking, each conformation creates unique hotspots that bind chemically distinguishable ligands. Clustering revealed insightful exceptions and the impact of crystal packing. Overall, Hsp90α’s plasticity provides a cautionary tale of overinterpreting individual crystal structures and motivates an ensemble-based view of drug design.