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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.

Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum. Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner. Here, we present high-resolution cryo–electron microscopy structures of human OST-A and OST-B. Although they have similar overall architectures, structural differences in the catalytic subunits STT3A and STT3B facilitate contacts to distinct OST subunits, DC2 in OST-A and MAGT1 in OST-B. In OST-A, interactions with TMEM258 and STT3A allow ribophorin-I to form a four-helix bundle that can bind to a translating ribosome, whereas the equivalent region is disordered in OST-B. We observed an acceptor peptide and dolichylphosphate bound to STT3B, but only dolichylphosphate in STT3A, suggesting distinct affinities of the two OST complexes for protein substrates.

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. The structure of human ABCG2 bound to an inhibitory antibody using cryo-electron microscopy, representing the first high-resolution structural data of a human multidrug transporter. Structure of a human multi-drug transporter ABCG2 is an ATP-binding cassette (ABC) transporter sometimes referred to as the breast cancer resistance protein. It is a multi-drug transporter that protects many tissues from foreign molecules and as such it has a key role in the effectiveness of commonly used oral drugs. It is also important in cancer treatments for the potential exploitation of the delivery of therapeutics to tumour cells. Here the authors present the structure of human ABCG2 bound to an inhibitory antibody, obtained using cryo-electron microscopy. This represents the first high-resolution structural data of a human multi-drug transporter. Two cholesterol molecules are bound to the multidrug binding pocket between the transmembrane domains, providing key information on substrate recognition by other ABC transporters.

ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood–brain, blood–testis and maternal–fetal barriers 1 – 4 . Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects 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 its inhibition by small molecules and antibodies 13 , 14 . However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2 EQ ), 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-sulfate (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-bound state, the substrate-binding cavity has 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, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a ‘plug’ between the two cavities. Our results show 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 for distinguishing substrates from inhibitors. Cryo-electron microscopy structures of the ABCG2 protein in ATP- and substrate-bound states reveal the location of substrate binding, conformational changes required for substrate translocation and how inhibitors might be distinguished from substrates.

The organic anion transporting polypeptides OATP1B1 and OATP1B3 are membrane proteins that mediate uptake of drugs into the liver for subsequent conjugation and biliary excretion, a key step in drug elimination from the human body. Polymorphic variants of these transporters can cause reduced drug clearance and adverse drug effects such as statin-induced rhabdomyolysis, and co-administration of OATP substrates can lead to damaging drug-drug interaction. Despite their clinical relevance in drug disposition and pharmacokinetics, the structure and mechanism of OATPs are unknown. Here we present cryo-EM structures of human OATP1B1 and OATP1B3 bound to synthetic Fab fragments and in functionally distinct states. A single estrone-3-sulfate molecule is bound in a pocket located in the C-terminal half of OATP1B1. The shape and chemical nature of the pocket rationalize the preference for diverse organic anions and allow in silico docking of statins. The structure of OATP1B3 is determined in a drug-free state but reveals a bicarbonate molecule bound to the conserved signature motif and a histidine residue that is prevalent in OATPs exhibiting pH-dependent activity. A key step of drug metabolism in the human body is the uptake into liver cells, which is mediated by transport proteins of the OATP family. Here, authors report cryo-EM structures of two human OATP proteins, providing insight into their function.

The distribution of relaxation times (DRT) analysis of impedance spectra is a proven method to determine the number of occurring polarization processes in lithium-ion batteries (LIBs), their polarization contributions and characteristic time constants. Direct measurement of a spectrum by means of electrochemical impedance spectroscopy (EIS), however, suffers from a high expenditure of time for low-frequency impedances and a lack of general availability in most online applications. In this study, a method is presented to derive the DRT by evaluating the relaxation voltage after a current pulse. The method was experimentally validated using both EIS and the proposed pulse evaluation to determine the DRT of automotive pouch-cells and an aging study was carried out. The DRT derived from time domain data provided improved resolution of processes with large time constants and therefore enabled changes in low-frequency impedance and the correlated degradation mechanisms to be identified. One of the polarization contributions identified could be determined as an indicator for the potential risk of plating. The novel, general approach for batteries was tested with a sampling rate of 10 Hz and only requires relaxation periods. Therefore, the method is applicable in battery management systems and contributes to improving the reliability and safety of LIBs.

The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar. Our structure reveals the transporter in an occluded conformation with a central, enclosed, inhibitor-binding pocket lined by residues from all transmembrane (TM) helices of ABCB1. The pocket spans almost the entire width of the lipid membrane and is occupied exclusively by two closely interacting zosuquidar molecules. The external, conformational epitope facilitating UIC2 binding is also visualized, providing a basis for its inhibition of substrate efflux. Additional cryo-EM structures suggest concerted movement of TM helices from both halves of the transporters associated with closing the NBD gap, as well as zosuquidar binding. Our results define distinct recognition interfaces of ABCB1 inhibitory agents, which may be exploited for therapeutic purposes.

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.

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.Cryo-EM structure of human transporter ABCB4 that extrudes phosphatidylcholine into the bile canaliculi suggests an ‘alternating access’ mechanism of lipid extrusion, distinct from the ‘credit card swipe’ model of other lipid transporters.

ABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of small-molecule inhibitors or inhibitory antibodies. Here we present cryo-EM structures of ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We find two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible substrate cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded substrate cavity. Substrate size appears to control which turnover state is mainly populated. The conformational changes between turnover-1 and turnover-2 states reveal how ATP binding is linked to the closing of the cytoplasmic side of the TMDs. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs. ABCG2 is a transporter contributing to multidrug resistance of cancer cells. Here, structures of human ABCG2 under turnover conditions reveal distinct conformational states, provide insight into the transport cycle and suggest a mechanism of discrimination between substrates and inhibitors.