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.

CAAX proteins play essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis 1. The ~120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases 2. Proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of their C-terminal CAAX motifs 3 (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve cysteine prenylation, -AAX tripeptide cleavage, and methylation of the carboxyl prenylated Cys residue. The major CAAX protease activity is mediated by the Ras and a-factor converting enzyme 1 (Rce1), an integral membrane protease of the endoplasmic reticulum 4,5. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here, we report the crystal structure of a Methanococcus maripaludis homolog of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site, are distinct from other intramembrane proteases (IMPs). Catalytic residues are located ~10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. The farnesyl lipid is proposed to bind to a site at the opening of two transmembrane α-helices, which then positions the scissile bond adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.

The La protein is an important component of ribonucleoprotein complexes that acts mainly as an RNA chaperone to facilitate correct processing and maturation of RNA polymerase III transcripts, but can also stimulate translation initiation. We report here the structure of the C-terminal domain of human La, which comprises an atypical RNA recognition motif (La225-334) and a long unstructured C-terminal tail. The central beta sheet of La225-334 reveals novel features: the putative RNA binding surface is formed by a five-stranded beta sheet and, strikingly, is largely obscured by a long C-terminal alpha helix that encompasses a recently identified nuclear retention element. Contrary to previous observations, we find that the La protein does not contain a dimerization domain.