Structure of the human multidrug transporter ABCG2

Abstract

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.

Tyrphostin RG14620 selectively reverses ABCG2-mediated multidrug resistance in cancer cell lines

“…For identification of the potential site(s) where tyrphostin RG14620 interacts with the substrate-binding pocket of ABCG2, docking studies of tyrphostin RG14620 were performed on 7 possible binding cavities within the transmembrane domain (TMD) of the recently published cryo-EM structure of a homodimer of the human ABCG2 transporter [54]. The best ligand binding pose (CDOCKER interaction energy = -26.6 kcal/mol) lies in a site lined by Val 401 , Thr 402 , Val 404 from transmembrane helix 1b (TM1b of monomer 2), Phe 432 , Asn 436 , Thr 435 , Phe 439 from TM2 and Met 549 from TM5 of monomer 1 of the dimer (Fig.…”

“…The energy was minimized for both the ABCG2 protein structure (PDB:5NJG) [54] and tyrphostin RG14620 using Acclerys Discovery Studio 4.0. Ligand preparation and docking was performed by the CDOCKER module of the same software and the ligand poses were ranked using the CDOCKER interaction energy parameters (kcal/mol).…”

Cryo-EM sample preparation method for extremely low concentration liposomes

“…One method is to use lipid nanodiscs, where the membrane protein resides in a small patch of lipid bilayer encircled by an amphipathic scaffolding protein (Bayburt et al, 2002). The nanodisc method has been employed to study the anthrax toxin pore at 22-Å resolution (Katayama et al, 2010), TRPV1 ion channel in complex with ligands at 3-4 Å resolution (Gao et al, 2016), and other membrane proteins (Autzen et al, 2018;Bayburt et al, 2002;Chen et al, 2017;Dang et al, 2017;Gao et al, 2016;Jackson et al, 2018;Katayama et al, 2010;McGoldrick et al, 2018;Roh et al, 2018;Srivastava et al, 2018;Taylor et al, 2017). Another method, called "random spherically constrained" (RSC) single-particle cryo-EM, where the membrane protein is reconstituted into liposomes, was developed and employed to study the large conductance voltage-and calcium-activated potassium (BK) channels reconstituted into liposomes at 17-Å resolution (Wang and Sigworth, 2009).…”

Structure‐guided probing of the leukotriene C4binding site in human multidrug resistance protein 1 (MRP1; ABCC1)

“…Because of their large size and hydrophobicity, it has been exceptionally challenging to acquire high-resolution atomic structures of mammalian ABC transporters by X-ray crystallography (17). However, this hurdle may be substantially overcome by recent advances in cryoelectron microscopy (cryo-EM) (18)(19)(20). Three cryo-EM structures of bovine Mrp1 (bMrp1) have been published and include substrate-free ("apo") bMrp1, LTC 4 -bound bMrp1, and nucleotide-bound mutant bMrp1-ATP-E1454Q (21,22).…”