Nucleotide binding is the critical regulator of ABCG2 conformational transitions

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• ABCG2 is an ATP-binding cassette (ABC) transporter.
• ABCG2 effluxes xenobiotics and endobiotics, contributing to multidrug resistance in cancer.
• The precise mechanisms of substrate translocation and ATP hydrolysis by ABCG2 are not fully understood.

Purpose of the Study:

• To investigate substrate and nucleotide binding by ABCG2 in cells.
• To elucidate the conformational changes of ABCG2 during its transport cycle.
• To understand the relationship between drug binding, nucleotide binding, and transporter conformation.

Main Methods:

• Development of methods to study substrate and nucleotide binding by ABCG2 concurrently.
• Utilizing a conformation-sensitive antibody (5D3) to monitor ABCG2 conformational states.
• Measuring antibody and substrate binding affinities to ABCG2.

Main Results:

• Nucleotide binding induces the transition of ABCG2 from an inward-facing (IF) to an outward-facing (OF) conformation.
• Substrates facilitate the IF-to-OF transition, while the inhibitor Ko143 hinders it.
• A high-to-low affinity switch in the drug-binding site accompanies the IF-to-OF conformational change.
• Low substrate binding is maintained in the post-hydrolysis state, indicating product dissociation is necessary for resetting the IF conformation.

Conclusions:

• Nucleotide binding and substrate interaction are key regulators of ABCG2 conformation and function.
• The conformational cycle of ABCG2, involving IF and OF states, is tightly linked to substrate binding and release.
• Understanding these molecular details can inform strategies to overcome chemotherapy resistance mediated by ABCG2.