bilirubin ditaurate  (Frontier Specialty Chemicals Inc)

Journal: Nature Communications

Article Title: Transport mechanism of human bilirubin transporter ABCC2 tuned by the inter-module regulatory domain

doi: 10.1038/s41467-024-45337-5

Figure Lengend Snippet: a The ATPase activities of ABCC2 wild type (WT) and E1462Q mutant. The data points of WT activity are fitted with the Michaelis-Menten equation. b The substrate-stimulated ATPase activity of ABCC2 upon the addition of conjugated bilirubin analog BDT. The data points are fitted with the Hill equation. All data points of ( a ) and ( b ) represent means of independent experiments ( n = 3) and the error bars indicate the means ± standard deviation (SD). c The refined cryo-EM maps of three ABCC2 structures. The unsharpened maps are displayed as the outline to show the position of detergent micelle. The cryo-EM maps are colored by UCSF ChimeraX 1.5 according to the local resolution estimated by cryoSPARC 3.1.

Article Snippet: To measure the ATPase activities of ABCC2 against different bilirubin ditaurate (BDT, disodium salt) (GC42931-10, GLPBIO) or estradiol-17β-D-glucuronide (E 2 17βG, disodium salt) (GC10964-10, GLPBIO) concentrations or varying ATP concentrations, protein at a final concentration of 0.05 μM was added to 75 μL reaction buffer containing 50 mM Tris-HCl, pH 7.4, 50 mM KCl, 1 mM DTT, 0.06% (w/v) digitonin, 2 mM MgCl 2 .

Techniques: Mutagenesis, Activity Assay, Standard Deviation, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Transport mechanism of human bilirubin transporter ABCC2 tuned by the inter-module regulatory domain

doi: 10.1038/s41467-024-45337-5

Figure Lengend Snippet: a Cartoon representation of the apo-form ABCC2. The TMD1&NBD1 module is colored in slate, the TMD2&NBD2 module is colored in lightpink, TMD0 is colored in wheat, the lasso motif is colored in meitnerium and the R domain is colored in green, respectively. The unmodeled regions of the R domain are represented by dashed lines. The apical canalicular membrane of hepatocytes is indicated as the gray lines. b Top view of cartoon representation of the TMDs. The transmembrane helices (TMs) are sequentially numbered. c Topological diagram of ABCC2 is colored using the same color scheme as shown in ( a ). Terminal residues of each structural segments are labeled. The TMs and coupling helices (CH) are sequentially numbered. The membrane plane is indicated as the gray rectangle. d The interactions between the R domain and TMDs. Interacting residues are shown as sticks. Carbon atoms are colored consistent with domain colors in ( a ), with oxygen in red and nitrogen in blue. Hydrogen bonds and salt bridges are shown as black dotted lines, with the distance in Å. e The ATPase activities of ABCC2 WT and two mutants in the R domain. The data are fitted using the Michaelis-Menten equation. Each data point is the average of three independent experiments ( n = 3), and error bars represent the means ± SD. f The transport activity assays of ABCC2 and two mutants in the R domain, using radioisotope-labeled substrate E 2 17βG. The transport activities of mutants are normalized by WT. Each data point is the average of independent experiments ( n = 3), and error bars represent the means ± SD. One-way analysis of variance (One-way ANOVA) is used for the comparison of statistical significance. The p value of E892Q is <0.0001, and the p value of E893Q is 0.0007. The P values of <0.05, 0.01, and 0.001 are indicated with *, **, and ***, respectively. Source data are provided as a Source Data file.

Article Snippet: To measure the ATPase activities of ABCC2 against different bilirubin ditaurate (BDT, disodium salt) (GC42931-10, GLPBIO) or estradiol-17β-D-glucuronide (E 2 17βG, disodium salt) (GC10964-10, GLPBIO) concentrations or varying ATP concentrations, protein at a final concentration of 0.05 μM was added to 75 μL reaction buffer containing 50 mM Tris-HCl, pH 7.4, 50 mM KCl, 1 mM DTT, 0.06% (w/v) digitonin, 2 mM MgCl 2 .

Techniques: Membrane, Labeling, Activity Assay, Comparison

Journal: Nature Communications

Article Title: Transport mechanism of human bilirubin transporter ABCC2 tuned by the inter-module regulatory domain

doi: 10.1038/s41467-024-45337-5

Figure Lengend Snippet: a Side and ( b ) top view of the overall structure of BDT-bound ABCC2 colored using the same color scheme as shown in Fig. . The BDT and cholesterol (CHL) molecules are shown as orange and yellow spheres, respectively. c Zoom-in view of the BDT binding site. The BDT molecule, CHL molecule and the interacting residues of ABCC2 are shown as sticks. Carbon atoms are colored consistent with domain colors in ( a ), with oxygen in red, nitrogen in blue and sulfur in yellow. Hydrogen bonds and salt bridges are shown as black dotted lines, with the distances in Å. d Superposition of the apo-form against the BDT-bound ABCC2. The zoom-in image on the right shows the superposition of the R domain and BDT/CHL molecules. e Superposition of the shared residues interacting with the R domain and BDT/CHL molecules from the apo-form (gray) and BDT-bound structure (slate), respectively. f Substrate-stimulated ATPase activities of ABCC2 WT and mutants that harboring a single mutation of residues at the substrate-binding pocket. Each data point is the average of independent experiments ( n = 3), and error bars represent the means ± SD. One-way ANOVA is used for the comparison of statistical significance of WT and mutants. The p values of all mutants are <0.0001. The P values of <0.05, 0.01, and 0.001 are indicated with *, **, and ***, respectively. Source data are provided as a Source Data file.

Article Snippet: To measure the ATPase activities of ABCC2 against different bilirubin ditaurate (BDT, disodium salt) (GC42931-10, GLPBIO) or estradiol-17β-D-glucuronide (E 2 17βG, disodium salt) (GC10964-10, GLPBIO) concentrations or varying ATP concentrations, protein at a final concentration of 0.05 μM was added to 75 μL reaction buffer containing 50 mM Tris-HCl, pH 7.4, 50 mM KCl, 1 mM DTT, 0.06% (w/v) digitonin, 2 mM MgCl 2 .

Techniques: Binding Assay, Mutagenesis, Comparison

Journal: Nature Communications

Article Title: Transport mechanism of human bilirubin transporter ABCC2 tuned by the inter-module regulatory domain

doi: 10.1038/s41467-024-45337-5

Figure Lengend Snippet: a Side and ( b ) top view of the overall structure of ATP/ADP-bound ABCC2 with the same color scheme as in Fig. . The ATP and ADP molecules are shown as yellow sticks, and Mg 2+ is shown as a green sphere. c Conformational changes upon ATP binding, shown as a cutaway representation of the electrostatic surface. TM8/TM9/TM14/TM15 are shown as cartoon. The electrostatic surface was generated by PyMOL 2.5.2 ( https://pymol.org ). d A detailed view of interface between the R domain and TMD2/NBD1. All interacting residues are shown as sticks. Hydrogen bonds and salt bridges are shown as black dotted lines, with the distances in Å. e The BDT- and ( f ) E 2 17βG-stimulated ATPase activity assays of ABCC2 and the R1150H mutant. The data points are fitted with the Hill equation in ( e ) and the Michaelis-Menten equation in ( f ). All data points for ( e ) and ( f ) represent means of three independent measurements ( n = 3), and error bars represent the means ± SD. g The transport activity assays of ABCC2 the R1150H mutant using radioisotope-labeled substrate E 2 17βG. The transport activities of mutants are normalized by WT. Each data point is the average of independent experiments ( n = 3), and error bars represent the means ± SD. One-way ANOVA is used for the comparison of statistical significance. The p value of R1150H is 0.0085. The P values of <0.05, 0.01, and 0.001 are indicated with *, **, and ***, respectively. Source data are provided as a Source Data file.

Article Snippet: To measure the ATPase activities of ABCC2 against different bilirubin ditaurate (BDT, disodium salt) (GC42931-10, GLPBIO) or estradiol-17β-D-glucuronide (E 2 17βG, disodium salt) (GC10964-10, GLPBIO) concentrations or varying ATP concentrations, protein at a final concentration of 0.05 μM was added to 75 μL reaction buffer containing 50 mM Tris-HCl, pH 7.4, 50 mM KCl, 1 mM DTT, 0.06% (w/v) digitonin, 2 mM MgCl 2 .

Techniques: Binding Assay, Generated, Activity Assay, Mutagenesis, Labeling, Comparison

Journal: Nature Communications

Article Title: Transport mechanism of human bilirubin transporter ABCC2 tuned by the inter-module regulatory domain

doi: 10.1038/s41467-024-45337-5

Figure Lengend Snippet: A schematic illustration of the transport cycle of ABCC2. The apo-form ABCC2 adopts an inward-facing conformation, with M-segment of the R domain in the substrate-binding pocket. The N-segment is identified as a loop in the apo form, whereas the C-segment is missing. The M-segment is expelled upon substrate binding, resulting in the missing of R domain in this structure. The ATP binding and hydrolysis at consensus site facilitate the substrate release, meanwhile, the C-segment of the R domain docks to the lateral of TMD2, which might maintain this active turnover state. Finally, the release of nucleotides accompanied with dissociation of the NBD dimer resets ABCC2 to the rest state. TMD0 and the two half modules of TMD1&NBD1 and TMD2&NBD2 are colored with the same color scheme as Fig. . The N-segment of the R domain is colored in blue, the M-segment is colored in green and the C-segment is colored in red, respectively. The untraceable regions of the R domain are represented by dashed lines.

Article Snippet: To measure the ATPase activities of ABCC2 against different bilirubin ditaurate (BDT, disodium salt) (GC42931-10, GLPBIO) or estradiol-17β-D-glucuronide (E 2 17βG, disodium salt) (GC10964-10, GLPBIO) concentrations or varying ATP concentrations, protein at a final concentration of 0.05 μM was added to 75 μL reaction buffer containing 50 mM Tris-HCl, pH 7.4, 50 mM KCl, 1 mM DTT, 0.06% (w/v) digitonin, 2 mM MgCl 2 .

Techniques: Binding Assay