Fig. 5

AHSA1-K137 is identified as the site-specific targeting of Bufalin and KU-177. A hHsp90α-hAHSA1 complex model. a Overview of hHsp90α (left) interacted with hAHSA1 (right, colored in yellow). Both hHsp90α and hAHSA1 were represented in colored cartoon and corresponding transparent surface. The N-terminal (15-287), linker (288-323), middle domain (324-476), and C-terminal (477-698) domain of hHsp90α were colored in cyan, red, blue and green, respectively. b Detailed information of hHsp90α binding with hAHSA1. The key residues corresponding to hAHSA1 bound with hHsp90α were shown as orange and blue sticks, respectively. The key interactions between residues were depicted by red dotted lines. B Predicted binding modes of Bufalin targeting hAHSA1. Bufalin was shown in red sticks, and hAHSA1 was shown in yellow cartoon. Key residues were shown in gray sticks, and hydrogen bonds were depicted by dotted lines. Phenylalanine in mutation of AHSA1-N131F was shown in magenta sphere. C MST results of Bufalin on wild and site-directed mutagenesis (SDM) of hAHSA1. AHSA1-K137 was involved in hydrogen bond interactions with Bufalin, and this hydrogen bond interaction disappeared with mutation to alanine. D-E AHSA1-K137 was involved in hydrogen bond interactions with its co-chaperone, HSP90, and AHSA1/HSP90 client protein, CDK6 and PSMD2, and this hydrogen bond interaction disappeared while mutation to alanine or double mutation K137A/N131F, as demonstrated by Co-IP using HA antibody as bait in ARP1 HSP90-OE cells followed by WB. AHSA1-OE plasmid was linked with HA tag, while HSP90-OE plasmid was linked with FLAG tag. F The chemical structure of KU-177. G Structural details of the predicted binding modes of KU-177. KU-177 was shown in green sticks, and key residues of hHsp90α (green cartoon) and AHSA1 (yellow cartoon) were shown in orange and gray sticks, respectively. Conformational changes from a hHsp90α-binding conformation (blue transparent cartoon) to a KU-177 “Induced Fit” conformation (yellow cartoon), as well as corresponding residues, K137 and N131, were also presented. H Predicted binding modes of KU-177 targeting hAHSA1. KU-177 was shown in green sticks, and hAHSA1 was shown in yellow cartoon. Key residues were shown in gray sticks, and hydrogen bonds were depicted by dotted lines. Phenylalanine in mutation of N131F was indicated in magenta sphere. I MST results of KU-177 on wild and site-directed mutagenesis (SDM) of hAHSA1. K137 was involved in hydrogen bond interactions with KU-177, and this hydrogen bond interaction would disappear with mutation to alanine