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 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.