Structural basis of bile salt extrusion and small-molecule inhibition in human BSEP.

BSEP (ABCB11) is an ATP-binding cassette transporter that is expressed in hepatocytes and extrudes bile salts into the canaliculi of the liver. BSEP dysfunction, caused by mutations or induced by drugs, is frequently associated with severe cholestatic liver disease. We report the cryo-EM structure of glibenclamide-bound human BSEP in nanodiscs, revealing the basis of small-molecule inhibition. Glibenclamide binds the apex of a central binding pocket between the transmembrane domains, preventing BSEP from undergoing conformational changes, and thus rationalizing the reduced uptake of bile salts. We further report two high-resolution structures of BSEP trapped in distinct nucleotide-bound states by using a catalytically inactivated BSEP variant (BSEPE1244Q) to visualize a pre-hydrolysis state, and wild-type BSEP trapped by vanadate to visualize a post-hydrolysis state. Our studies provide structural and functional insight into the mechanism of bile salt extrusion and into small-molecule inhibition of BSEP, which may rationalize drug-induced liver toxicity.

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.