Identification of important binding sites for artesunate with GBA protein (n = 3) (IMAGE)
Caption
(A) Two- and three-dimensional images of artesunate with the binding pocket that formed with the amino acids of GBA and their interacting conventional hydrogen bonds using AutoDock Vina, Ledock, and Schrödinger molecular docking. (B) The expression levels of the FLAG tag and GBA protein were detected by Western blotting. (C, D) Quantitative results for FLAG and GBA protein expression levels are shown. (E) GBA enzymatic activity was measured. (F) Cellular thermal shift assay (CETSA)-Western blotting experiment detected the thermal stability of wild-type GBA and mutant-type GBA (Y313G, E340R, N396R, Y313G/E340R/N396R) plasmid mutant proteins after treatment with 29.27 μM artesunate. (G) The CETSA curve and the thermal stability to reach the Tm 50 value were performed using GraphPad Prism software. (H–M) Detection for binding affinities of artesunate with wild-type GBA and mutant-type GBA (Y313G, E340R, N396R, Y313G/E340R/N396R) using microscale thermophoresis (MST) assay. (H) Dose–response curves for calculating binding affinities between artesunate and wild-type GBA and mutant-type GBA (Y313G, E340R, N396R, Y313G/E340R/N396R) by MST. (I) Artesunate with wide-type GBA. (J) Artesunate with mutant-type GBA (N396R). (K) Artesunate with mutant-type GBA (Y313G). (L) Artesunate with mutant-type GBA (E340R). (M) Artesunate with mutant-type GBA (Y313G/E340R/N396R). WT-DMSO: wide-type GBA and DMSO group. WT-ART: wide-type GBA and 29.27 μM artesunate treatment group. Y313G-E340R–N396R-ART, E340R-ART, Y313G-ART, and N396R-ART: mutant-type GBA (Y313G/E340R/N396R, E340R, Y313G, N396R).
Credit
Xia Mao, Xiangying Yan, Yawen Chen, Bingbing Cai, Wenjia Chen, Ya Lin, Na Lin, Yanqiong Zhang
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CC BY-NC-ND