Nucleotide binding is the critical regulator of ABCG2 conformational transitions

Abstract

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.

A time-resolved Förster resonance energy transfer assay to investigate inhibitor binding to ABCG2

“…Structurally, the outward facing conformation of ABCG2 has a much narrower binding cavity resulting in the loss of specific interactions known to be vital for transport (figure 5c comparison of cavity 1 in the nucleotide free and bound structures of ABCG2). Our demonstration of a 10-fold reduction in affinity for Ko143-X- BY630 in the AMP-PNP bound state provides more evidence for the hypothesis that ATP binding is responsible for affinity switches in ABCG2[31, 32] and thus that ATP hydrolysis is responsible for transporter resetting to the inward facing conformation.…”

“…Having tools to explore the binding of substrates to transporters would improve the understanding of their structure-activity relationships, and the broader role of transporters in pharmacokinetics and metabolism. The present assay contributes to the tools available for studying ABCG2 and could be broadened to other MDR pumps [20, 31, 38].…”

Identification and immunoinfiltration analysis of key genes in ulcerative colitis using WGCNA

“…Subsequently, through analysis of the key genes, ABCG2, IL1RN, REG4, SERPINB5 and TRIM29 were finally screened out. ABCG2 (transporter) is an export ABC protein that can expel a wide range of chemically unrelated heterologous and endogenous substances from cells ( Gyongy et al, 2023 ). IL1RN(Interleukin-1 receptor antagonist gene) serves as a natural antagonist of IL-1.…”

ABCG2 in Acute Myeloid Leukemia: Old and New Perspectives

“…Transfer across the membrane is associated with conformation changes of the protein (“in-facing”, with the substrate binding site open in the cytoplasm, and “out-facing” open in the extracellular space). Recently, Gyöngy et al, and Yu et al, proposed two molecular models to explain drug transport, highlighting the crucial role of ATP binding to modulate ABCG2 conformation [ 86 , 87 ]. As for the other ABC members, the molecular basis of ABCG2 substrate specificity is not fully elucidated.…”