ABCB1/P-glycoprotein actively extrudes xenobiotic compounds across the plasma membrane of diverse cells, which contributes to cellular drug resistance and interferes with therapeutic drug delivery. We determined the 3.5Å cryo-EM structure of substrate-bound human ABCB1 reconstituted in lipidic nanodiscs, revealing a single molecule of the chemotherapeutic compound paclitaxel bound in a central, occluded pocket. A second structure of inhibited, human-mouse chimeric ABCB1 revealed two molecules of zosuquidar occupying the same drug-binding pocket. Minor structural differences between substrate-and inhibitor-bound ABCB1 sites are amplified towards the NBDs, revealing how the plasticity of the drug-binding site controls the dynamics of the ATP-hydrolyzing NBDs. Ordered cholesterol and phospholipid molecules suggest how the membrane modulates the conformational changes associated with drug binding and transport.
ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.
ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.
ABCG2 is a multidrug ATP-binding cassette transporter expressed in the plasma membranes of various tissues and tissue barrier [ 1 – 4 ]. It translocates endogenous substrates, affects the pharmacokinetics of many drugs, and has a protective role against a wide array of xenobiotics, including anti-cancer drugs [ 5 – 12 ]. Previous studies have revealed the architecture of ABCG2 and the structural basis of small-molecule and antibody inhibition [ 13 , 14 ], but the mechanism of substrate recognition and ATP-driven transport are currently unknown. Here we present high-resolution cryo-EM structures of human ABCG2 in two key states, a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, a mutant containing a glutamine replacing the catalytic glutamate (ABCG2 EQ ) was used, which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulphate (E 1 S) is bound in a central, hydrophobic, and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of E 1 S can bind in the observed binding mode. In the ATP-boundstate, the substrate-binding cavity has completely collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains (TMDs), pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis of residues contacting bound E 1 S or in the translocation pathway, followed by in vitro characterization of transport and ATPase activities, demonstrated their roles in substrate recognition and revealed the importance of a leucine residue forming a ‘plug’ between the two cavities. Our results reveal how ABCG2 harnesses the energy of ATP binding to extrude E 1 S and other substrates and suggest that the size and binding affinity of compounds are important parameters in distinguishing substrates from inhibitors.
ABCB1 detoxifies cells by exporting diverse xenobiotic compounds, thereby limiting drug disposition and contributing to multidrug resistance in cancer cells. Multiple small-molecule inhibitors and inhibitory antibodies have been developed for therapeutic applications, but the structural basis of their activity is insufficiently understood. We determined cryo-EM structures of nanodisc-reconstituted, human ABCB1 in complex with the Fab fragment of the inhibitory, monoclonal antibody MRK16 and bound to a substrate (the antitumor drug vincristine) or to the potent inhibitors elacridar, tariquidar, or zosuquidar. We found that inhibitors bound in pairs, with one molecule lodged in the central drug-binding pocket and a second extending into a phenylalanine-rich cavity that we termed the “access tunnel.” This finding explains how inhibitors can act as substrates at low concentration, but interfere with the early steps of the peristaltic extrusion mechanism at higher concentration. Our structural data will also help the development of more potent and selective ABCB1 inhibitors.
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