Structural Basis of Drug Recognition by the Multidrug Transporter ABCG2. Issue 13 (25th June 2021)

• Record Type: Journal Article
• Authors: Kowal, Julia; Ni, Dongchun; Jackson, Scott M.; Manolaridis, Ioannis; Stahlberg, Henning; Locher, Kaspar P.
• Is Part Of: Journal of molecular biology. Volume 433:Issue 13(2021)
• Publication Date: 2021-06-25
• Subjects: ABCG2 -- BCRP -- topotecan -- mitoxantrone -- tariquidar; ABC ATP-binding cassette -- E1S estrone-3-sulfate -- cryo-EM cryo-electron microscopy -- BCRP breast cancer resistance protein -- NBDs nucleotide binding domains -- TMDs transmembrane domains -- 3DVA 3D variability analysis; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2021.166980
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: Anticancer drugs interact with key amino acid residues in the binding pocket of ABCG2. Substrate retains some mobility inside the drug-binding pocket of ABCG2. Residues responsible for interactions with substrates were identified. Structural flexibility of NBDs is reduced by substrate binding. Abstract: ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the endogenous substrate estrone-3-sulfate (E1 S) has been elucidated at a structural level, the recognition and recruitment of exogenous compounds is not understood at sufficiently high resolution. Here we present three cryo-EM structures of nanodisc-reconstituted, human ABCG2 bound to anticancer drugs tariquidar, topotecan and mitoxantrone. To enable structural insight at high resolution, we used Fab fragments of the ABCG2-specific monoclonal antibody 5D3, which binds to the external side of the transporter but does not interfere with drug-induced stimulation of ATPase activity. We observed that the binding pocket of ABCG2 can accommodate a single tariquidar molecule in a C-shaped conformation, similar to one of the two tariquidar molecules bound to ABCB1, where tariquidar acts as an inhibitor. We also found single copies of topotecan and mitoxantrone bound between key phenylalanine residues. Mutagenesis experiments confirmed the functional importance of two residues in the binding pocket, F439 and N436. Using 3D variability analyses, we found a correlation between substrate binding and reduced dynamics of the nucleotide binding domains (NBDs), suggesting a structural explanation for drug-induced ATPase stimulation. Our findings provide additional insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates.