Fig. 2: Cryo-EM structure and functional analysis of glibenclamide-bound BSEP.

a Normalized ATPase activity of BSEP in detergent as a function of increasing concentration of TC or GBM. b Normalized inhibition of TC transport as a function of GBM concentration in the presence of 50.5 μM TC and 5 mM ATP. The points were plotted using nonlinear regression of the Michaelis-Menten equation and the IC₅₀ was calculated to be ~10 μM and the 95% confidence intervals (CI) range is 5–22 μM. c Chemical structure of GBM drawn in ChemDraw. d Cryo-EM density map and structure of BSEP in a complex with GBM (magenta). The right panels show GBM in two different orientations and the molecular interactions in the central cavity, shown together with the density of GBM (blue). Residues interacting with GBM (Q76, L80, F83, F776, N996 and S1022) are shown as sticks. e Comparison of GBM (this study) and TC binding sites (PDB 7E1A). f ATPase activity of BSEP and mutants (Q76A, L80F, F83A, F776A, N996A, and S1022F) in the presence or the absence of GBM. The increase of GBM-stimulated ATPase activity is indicated. 300 μM GBM was added and pre-incubated prior to the reaction. 5 mM ATP was used for initiating the assay. Data points in a, b, and f indicate the mean of three independent measurements and error bars indicate SD. Statistical significance in f was determined using ordinary one-way ANOVA, Tukey’s multiple comparison test. Differences between wild-type BSEP and mutants were depicted as ****P ≤ 0.0001.