Structure of the human lipid exporter ABCB4 in a lipid environment.

ABCB4 is an ATP-binding cassette transporter that extrudes phosphatidylcholine into the bile canaliculi of the liver. Its dysfunction or inhibition by drugs can cause severe, chronic liver disease or drug-induced liver injury. We determined the cryo-EM structure of nanodisc-reconstituted human ABCB4 trapped in an ATP-bound state at a resolution of 3.2 Å. The nucleotide binding domains form a closed conformation containing two bound ATP molecules, but only one of the ATPase sites contains bound Mg2+. The transmembrane domains adopt a collapsed conformation at the level of the lipid bilayer, but we observed a large, hydrophilic and fully occluded cavity at the level of the cytoplasmic membrane boundary, with no ligand bound. This indicates a state following substrate release but prior to ATP hydrolysis. Our results rationalize disease-causing mutations in human ABCB4 and suggest an 'alternating access' mechanism of lipid extrusion, distinct from the 'credit card swipe' model of other lipid transporters.

Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformation.

The multidrug transporter permeability (P)-glycoprotein is an adenosine triphosphate (ATP)-binding cassette exporter responsible for clinical resistance to chemotherapy. P-glycoprotein extrudes toxic molecules and drugs from cells through ATP-powered conformational changes. Despite decades of effort, only the structures of the inward-facing conformation of P-glycoprotein are available. Here we present the structure of human P-glycoprotein in the outward-facing conformation, determined by cryo-electron microscopy at 3.4-angstrom resolution. The two nucleotide-binding domains form a closed dimer occluding two ATP molecules. The drug-binding cavity observed in the inward-facing structures is reorientated toward the extracellular space and compressed to preclude substrate binding. This observation indicates that ATP binding, not hydrolysis, promotes substrate release. The structure evokes a model in which the dynamic nature of P-glycoprotein enables translocation of a large variety of substrates.

Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle.

The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP.

Molecular basis of the polyspecificity of P-glycoprotein (ABCB1): recent biochemical and structural studies.

ABCB1 (P-glycoprotein/P-gp) is an ATP-binding cassette transporter well known for its association with multidrug resistance in cancer cells. Powered by the hydrolysis of ATP, it effluxes structurally diverse compounds. In this chapter, we discuss current views on the molecular basis of the substrate polyspecificity of P-gp. One of the features that accounts for this property is the structural flexibility observed in P-gp. Several X-ray crystal structures of mouse P-gp have been published recently in the absence of nucleotide, with and without bound inhibitors. All the structures are in an inward-facing conformation exhibiting different degrees of domain separation, thus revealing a highly flexible protein. Biochemical and biophysical studies also demonstrate this flexibility in mouse as well as human P-gp. Site-directed mutagenesis has revealed the existence of multiple transport-active binding sites in P-gp for a single substrate. Thus, drugs can bind at either primary or secondary sites. Biochemical, molecular modeling, and structure-activity relationship studies suggest a large, common drug-binding pocket with overlapping sites for different substrates. We propose that in addition to the structural flexibility, the molecular or chemical flexibility also contributes to the binding of substrates to multiple sites forming the basis of polyspecificity.