Fig. 1: Topology and functional characterization of human BSEP.

a Topology diagram of human BSEP. TM helices (1–12), elbow helices, intracellular helices (IH1–IH4), and extracellular loops (ECL1–ECL6) are numbered and indicated. Stretches of amino acids not resolved in the structures are shown as dashed lines. NBS1 and NBS2 are colored yellow and pink, and the conserved motifs are indicated (A: Walker-A; B: Walker-B; S: Signature motif). The degenerate catalytic site is located in Walker-B labeled in red (B) of NBD1. Blue stars indicate regions that are in contact with GBM. b, c Normalized ATPase activity of BSEP and BSEP_E1244Q in detergent and proteoliposomes in the presence of substrate TC, GBM, and/or vanadate. d ATP-driven transport of ³H-TC by BSEP and BSEP_E1244Q in proteoliposomes. The observed TC transport rate of BSEP_E1244Q in the absence of ATP is considered background. 50.5 μM TC was added and incubated prior to the reaction. The reaction was started by adding ATP and the concentration of ATP was 5 mM throughout in (b–d). The data points above indicate means of three independent measurements and error bars indicate SD. Statistical significance was determined using ordinary one-way ANOVA, Tukey’s multiple comparison test. Differences between basal activity and stimulated activity or between transport activities in the presence or absence of ATP were depicted as ****P ≤ 0.0001 and ***P = 0.0001.