A structural and mechanistic model for BSEP dysfunction in PFIC2 cholestatic disease

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 f..., CETSA
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 1.2x106 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®).
Melting curve a..., , # **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...,