Structures of ABCB4 provide insight into phosphatidylcholine translocation

Abstract

ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposome-based translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs.

Keywords: ABC transporter; cryo-EM; hepatocyte; membrane transport; phosphatidylcholine.

Fig. 1.

Structure of wild-type ABCB4 and functional analysis. (A) Structures of apo-inward and occluded ABCB4 (Center), with TMDs shown as ribbons and NBDs as surfaces. The N- and carboxyl-terminal halves of ABCB4 are colored differently. Bound phosphatidylcholine is shown as purple spheres. (Left) Close-up of the apo-inward conformation shown as a surface representation. The side opening to the inner leaflet of the membrane is indicated by an orange arrow. (Right) Close-up of the occluded conformation. The central cavity, generated with HOLLOW (47), is shown as an orange surface. The phosphatidylcholine binding pocket is located above the residue W234, shown as blue sticks. The asterisk depicts the area termed the access tunnel in the related ABCB1 protein (16). (B) Binding pocket of PC molecule in occluded conformation. Bound PC and interacting residues are shown as sticks; EM density is shown as blue mesh. (C) Normalized PC extrusion rates from HEK293T cells expressing wild-type or mutant ABCB4. The results are normalized to the amount of ABCB4 extracted from the membranes relative to wild-type ABCB4. Means of measurements from at least three biological replicates are shown; error bars represent SDs. Statistical significance was determined using Dunnett’s test. Differences between wild-type and mutants were depicted as *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. (D) Normalized in vitro transport activity of wild-type ABCB4. Proteoliposomes contained liver polar lipid extract and cholesterol (4:1 wt/wt) supplemented with 0.3% (wt/wt) NBD-PC. Numbers refer to the alkyl tail lengths and the number of double bonds. Data were normalized to the highest transport rate. Means of measurements from at least three independent proteoliposomes-based samples are shown; error bars represent SDs. Statistical significance was determined using Dunnett’s test. Differences between 14:0 and 6:0 NBD-PC condition and other samples were depicted as *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Fig. 2.

Structure of posaconazole-inhibited ABCB4. (A, Left) TMDs are shown as ribbons and NBDs as surfaces. The N- and carboxyl-terminal halves of ABCB4 are colored differently. Posaconazole and PC are shown in sphere representation and colored green and purple, respectively. (Right) Surface representation. The blue arrow shows the side opening to the membrane. (B) Horizontal slice through a ribbon representation, with posaconazole and PC shown as green and purple sticks. (C) Close-up views of the TMDs in distinct states. Bound PC and posaconazole are shown in sticks and colored as in B. The TM helices 4 and 10 undergoing conformational changes are shown as ribbons. Posaconazole prevents bound PC from reaching the central pocket and TM4 from adopting a kinked conformation.

Fig. 3.

Structural changes of ABCB4 during transport cycle. (A) Horizontal slices through the TMDs of available ABCB4 structures, shown from the inside of the cell. TM helices are shown as ribbons and numbered. Bound PC and posaconazole and the side chain of W234 are shown as sticks. The orange arrow shows the side entrance from the inner leaflet of the membrane. (B) Proposed structure-based reaction mechanism. The N- and carboxyl-terminal halves of ABCB4 are colored yellow and blue. The red arch depicts the choline binding site including the side chain of W234. Numbers in yellow circles depict functionally relevant conformational states: States 1, 2, and 2′ represent structures from this study, state 4 is based on PDB: 6s7p (12), and state 3 is based on a putative outward-open conformation (12).