DOI: 10.5061/dryad.cnp5hqcgd
title: A structural and mechanistic model for BSEP dysfunction in PFIC2 cholestatic disease

description: BSEP (ABCB11) transports bile salts across the canalicular membrane of
 hepatocytes, where they are incorporated into bile. Biallelic mutations in
 BSEP can cause Progressive Familial Intrahepatic Cholestasis Type 2
 (PFIC2), a rare pediatric disease characterized by hepatic bile acid
 accumulation leading to hepatotoxicity and, ultimately, liver failure. The
 most frequently occurring PFIC2 disease-causing mutations are missense
 mutations, which often display a phenotype with decreased protein
 expression and impaired maturation and trafficking to the canalicular
 membrane. To characterize the mutational effects on protein thermodynamic
 stability, we carried out biophysical characterization of 13 distinct
 PFIC2-associated variants using in-cell thermal shift (CETSA)
 measurements. These experiments reveal a cluster of residues localized to
 the NBD2-ICL2 interface, which exhibit severe destabilization relative to
 wild-type BSEP. A high-resolution (2.8 Å) cryo-EM structure provides a
 framework for rationalizing the CETSA results, revealing a novel,
 NBD2-localized mechanism through which the most severe missense patient
 mutations drive cholestatic disease. These findings suggest potential
 strategies for identifying mechanism-based small molecule correctors to
 address BSEP trafficking defects and advance novel therapies for PFIC2 and
 other cholestatic diseases.
description: <strong>CETSA</strong> Adherent
 HEK293T cells were grown in a 6-well plate and transfected with BSEP-HiBiT
 constructs when they reached 70% confluency using PEI MAX® - Transfection
 Grade Linear Polyethyleneimine Hydrochloride (MW 40,000) (Polysciences) at
 a 1:3 DNA: PEI mass ratio. Cells were collected 24h after transfection by
 trypsinization, resuspended in fresh DMEM at a concentration of
 1.2x10<sup>6</sup> cells/ml, and dispensed in PCR tubes (20 μl
 per tube, 2 replicates per condition). Tubes were placed in a multi-block
 thermal cycler and submitted to a thermal cycle of 10 minutes
 pre-incubation at 37°C, 3.5 minutes heating step, and fast cooling to 4°C.
 Cells were then lysed, and a luminescent signal was generated by adding
 Nano-Glo® HiBiT Lytic Detection mix prepared per manufacturer’s
 instructions (Promega) to the heated samples at a 1:1 vol/vol ratio. Cell
 lysates were subsequently dispensed in a white 384 well plate (Corning)
 and luminescence was detected on a plate reader (TECAN Spark®).
 <strong>Melting curve analysis</strong>
 For each independent CETSA experiment, the luminescence of two
 wells per temperature were averaged. The resulting melting curve was
 fitted with a non-linear regression model with GraphPad Prism (Version 9,
 Boston, MA) from which a melting/aggregation temperature was extracted.
 Mean T<sub>agg </sub>values were obtained from at least three
 independent melting curves fitted individually (N=4 biological replicates
 for WT, R948C, R1128C, R1153C and R1268Q; N=3 for other
 variants). <strong>Protein
 purification</strong> A pellet of cells
 expressing WT BSEP or E297G mutant was resuspended in solubilization
 buffer (Tris 50 mM pH8, 150 mM NaCl, 10% glycerol, 2 mM DTT, 2 mM MgCl2,
 1%DDM, 0.2% CHS, protease inhibitor tablet and DNase/RNase) for 2 hours at
 4°C. The cell lysate was centrifuged at 200,000 g for 30 minutes at 4°C,
 and the supernatant was added to anti-GFP nanobody beads (Protein and
 Crystallography Facility, University of Iowa) for 1 hour at 4°C. Beads
 were washed with buffer A (150 mM NaCl, 50 mM Tris pH8, 2 mM DTT, 2 mM
 MgCl2, 0.02% GDN) and incubated with 3C protease (40 μM) for 3 hours at
 4°C to remove the C-terminal GFP tag. The eluted protein was further
 purified by gel filtration chromatography using a Superose® 6 Increase
 10/300 GL (Cytiva) equilibrated with Buffer A.
 <strong>Differential scanning fluorimetry 
 </strong> Freshly purified proteins were
 concentrated to ~0.4 mg/ml on a Pierce 100K MWCO concentrator (Thermo
 Fisher Scientific) catalog number 88503 for thermal stability analysis
 with Prometheus NT48 (NanoTemper Technologies). Tryptophan fluorescence
 330/350 nm ratio was measured in high sensitivity capillaries submitted to
 a temperature ramp of 1°C per minute, from 20 to 80 °C. Resulting melting
 curves were analyzed with PR.Stability Analysis software (NanoTemper).
 Melting temperatures were obtained from at least three independent
 experiments (N=3 for WT and N=6 for E297G).
 <strong>Activity assay and kinetic parameters for
 BSEP</strong> Purified BSEP activity was measured
 using the ADP-Glo assay format (Promega, Madison, WI) according to the
 vendor protocol. The assay buffer contained 25 mM Hepes, pH 8.5, 150 mM
 NaCl, 2 mM MgCl2, 2 mM DTT, 0.02% GDN, 0.002% Triton X-100, 0.001% faf
 BSA. Experimental conditions: Purified BSEP 100 nM, ATP 1 mM, Sodium
 Taurocholate (TCA): 1 – 0 mM (eight concentrations, 1:2
 dilution). 
description: # **A structural and mechanistic model for BSEP dysfunction in PFIC2
 cholestatic disease**
 [https://doi.org/10.5061/dryad.cnp5hqcgd](https://doi.org/10.5061/dryad.cnp5hqcgd) **Description of the data and file structure** The files contain raw data used in Cellular Thermal Shift Assay (CETSA) titrations (Figures 3 and 4), nanoDSF (Differential Scanning Fluorimetry) experiments (Figure 3) and characterization of the BSEP ATPase activity and kinetic parameters shown in Supplementary Figure 3. Excel files are provided for all data, and for convenience, GraphPad Prism files are also provided for the ATPase data. **Files and variables** **File: Figures_3_and_4_CETSA_raw_data.xlsx** **Description:** CETSA temperature titrations to determine the aggregation temperature of BSEP and 13 patient variants. In this experiment, the HiBiT split luciferase assay (Promega) was used to quantitate non-aggregated protein at each temperature studied. As the temperature is increased, protein will unfold and aggregate, resulting in a decrease in luminescence. The midpoint of the unfolding (aggregation) curve gives a value for Tagg. **Variables** * Temperature * Luminescence **File: Figure_3_NanoDSF_raw_data.xlsx** **Description:** nanoDSF melting experiments for WT and E297G BSEP. In this experiment, purified recombinant wild type BSEP and E297G variant proteins were subjected to a temperature challenge in a nanoDSF instrument (Prometheus NT48 from NanoTemper Technologies). For each temperature, Tryptophan fluorescence was measured at 330nm and 350nm. As the proteins unfold with increasing temperature, the fluorescence emission maximum shifts from 330nm to 350nm. Plotting the fluorescence ratio 350/330 as a function of temperature hence gives the protein’s unfolding profile (data shown in Figure 3B) from which a melting temperature (Tm) is obtained. Plotting the first derivative of the 350/330 ratio allows a better visualization of the unfolding midpoint (data shown in Figure 3C). The file provides the 350/330 fluorescence ratio and first derivative values for each replicate (N=3 for WT, N=6 for E297G), as well as the average of all replicates merged with the PR.Stability Analysis software (NanoTemper Technologies) for each protein as plotted in figure 3B and 3C. **Variables** * Temperature * Fluorescence **File: Enzyme_activity_assays_Prism_RCP006_22_TCA_Km_30m.pzfx** **Description:** Time dependence of ADP production. The assay format used in all enzyme activity assay data sets is the ADP-Glo assay from Promega (Madison, WI). In this assay, the amount of ATP converted into ADP is proportional to the measured luminescence. The luminescence is then interpolated versus a standard curve of luminescence vs ATP/ADP ratio to determine the amount of ADP produced. Supplementary Figure 3A shows the background-corrected data in the tab “ABCB11 kinetic subtracted” in the left panel and the interpolated, background-corrected data in the right panel from graph tab “Baseline-corrected of interpolation uM”. The Michaelis-Menten curve for TCA in panel B plotted in the “Velocity vs TCA molar” tab. **Variables for Supplementary Figure 3A left panel** * deltaRLU (relative to no TCA) (background-corrected luminescence) * Time **Variables for Supplementary Figure 3A right panel** * ADP produced * Time **Variables for Supplementary Figure 3B** * Reaction velocity * TCA concentration **File: Enzyme_activity_assays_RCP006_22_TCA_Km_30m.xlsx** **Description:** This file contains only raw luminescence data used in determining ABCB11 kinetics in Supplementary Figure 3A. “End point” and “Interpolation” tabs contain luminescence data before background correction and the interpolated luminescence data before background correction. **Variables (Endpoint tab)** * Luminescence * Time **Variables (Interpolation tab)** * Luminescence * Time **File: Enzyme_activity_assays_RCP006_22_ATP_Km.pzfx** **Description:** Determination of Km for ATP (GraphPad Prism format). This data is shown is Supplementary Figure 3. The panel showing dose dependence on ATP concentration is a plot of data in the Data 1 tab, and is shown in the Graph Data 1 tab. **Variables for Dose-dependence on ATP concentration** * Luminescence * ATP concentration **Variables for Michaelis-Menten curve for ATP** * Reaction velocity * ATP concentration **File: Enzyme_activity_assays_RCP006_22_ATP_Km.xlsx** **Description:** Determination of Km for ATP (Excel format). Description is same as for the Prism file listed above. **Variables for Dose-dependence on ATP concentration** * Luminescence * ATP concentration **Variables for Michaelis-Menten curve for ATP** * Reaction velocity * ATP concentration **Code/software** Microsoft Excel (Mac 16.94) GraphPad Prism (Version 10.4.1)