Structural analysis of de novo-designed binders targeting the closed state of HSP90

Heat shock protein 90 (HSP90) assists the folding and maturation of numerous signaling proteins and is an important therapeutic target. Most HSP90 inhibitors target the ATP-binding pocket and disrupt ATP-induced closure, whereas inhibitors targeting the closed state have not been developed. Here, we report de novo-designed protein binders that selectively recognize closed HSP90. Two binders, H2 and H4, have similar folds and affinities for HSP90 but distinct binding kinetics. Cryo-EM structures of HSP90 complexes with H2 and H4 revealed high accuracy of the BindCraft predictions. H2 and H4 compete with p23 at one, but not both, symmetrical p23-binding sites. In HEK293T cells, H2 induced a reduced heat shock response compared with the ATP-site inhibitor 17-DMAG while reducing levels of CDK4, providing functional evidence for HSP90 inhibition. These binders provide tools for probing HSP90 function and potential leads for developing closed-state HSP90 inhibitors.