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Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
08:33

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

Published on: March 11, 2021

Is ATP binding responsible for initiating drug translocation by the multidrug transporter ABCG2?

Christopher A McDevitt1, Emily Crowley, Gemma Hobbs

  • 1Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, UK.

The FEBS Journal
|July 29, 2008
PubMed

Insights

Drug binding to ABCG2 transporters initiates a conformational change, enabling chemotherapy efflux. Nucleotide binding, not hydrolysis, drives the transporter's "power stroke" for drug translocation.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • ABCG2 (ATP-binding cassette sub-family G member 2) is a transporter protein.
  • It confers cancer cell resistance by effluxing chemotherapeutic drugs.
  • ABCG2 possesses interconnected drug binding sites, influencing its function.

Purpose of the Study:

  • To investigate the coupling mechanism between drug binding and ATP hydrolysis in ABCG2.
  • To elucidate the role of nucleotide binding in the transporter's conformational changes and drug translocation.

Main Methods:

  • Utilized [3H]Daunomycin binding assays.
  • Examined the ABCG2 R482G isoform in nucleotide-bound and post-hydrolytic states.
  • Assessed the effect of ATP analogues on drug binding.

Main Results:

  • Initial drug binding involves a high-affinity interaction, transitioning to a low-affinity state.
  • ATP analogues displaced [3H]Daunomycin, indicating a pre-hydrolysis transition to low affinity.
  • The low-affinity state persisted immediately post-ATP hydrolysis, suggesting phosphate/ADP dissociation resets the transporter.

Conclusions:

  • Nucleotide binding, rather than hydrolysis, is the primary driver ('power stroke') for drug translocation in ABCG2 R482G.
  • This mechanism is similar to other ABC transporters like ABCB1 and ABCC1.
  • Understanding these dynamics is crucial for developing strategies to overcome chemotherapy resistance.

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