RT Journal Article
SR Electronic
T1 Nucleotide binding is the critical regulator of ABCG2 conformational transitions
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.10.19.512890
DO 10.1101/2022.10.19.512890
A1 Zsuzsanna Gyöngy
A1 Gábor Mocsár
A1 Éva Hegedűs
A1 Thomas Stockner
A1 Zsuzsanna Ritter
A1 László Homolya
A1 Anita Schamberger
A1 Tamás I. Orbán
A1 Judit Remenyik
A1 Gergely Szakács
A1 Katalin Goda
YR 2022
UL http://biorxiv.org/content/early/2022/10/21/2022.10.19.512890.abstract
AB ABCG2 is an exporter type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy.Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.Competing Interest StatementThe authors have declared no competing interest.A647Alexa 647 succinimidyl esterABCPPlacenta-Specific ABC transporterADME-Toxabsorption, distribution, metabolism, excretion and toxicityAMP-PNPadenylyl-imidodiphosphateBCRPBreast Cancer Resistance ProteinE3Sestrone-3-sulfatecryo-EMcryogenic electron microscopyCFTRCystic Fibrosis Transmembrane Conductance Regulator (ABCC7)FCSfluorescence correlation spectroscopyFBSfoetal bovine serumGFPgreen fluorescent proteinIFinward-facingmAbmonoclonal antibodyMXmitoxantroneMXRMitoxantrone Resistance ProteinNBDnucleotide binding domainOFoutward-facingPIpropidium iodideSLOstreptolysin-OTMDtransmembrane domainVivanadate