Cryogenic-electron microscopy (Cryo-EM) structures of a human ABCG2 mutant transporter protein

phys.org | 11/21/2018 | Staff
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The transporter protein ABCG2 belongs to the ATP-binding-cassette (ABC) family. The protein is expressed in the plasma membranes of cells within a variety of tissues and tissue barriers, including the blood-brain, blood-testes and maternal-fetal barrier. The protein can be powered by ATP to translocate endogenous substrates, affect the pharmacokinetics of many drugs and protect against a variety of xenobiotics including anticancer drugs, notably in breast cancer. ABCG2 is often referred to as the breast cancer resistance protein, where previous studies have revealed the ABCG2 architecture and structural basis of ABCG2 inhibition with small molecules and antibodies. The mechanism of substrate recognition by ABCG2 alongside its ATP-driven transport capability remains yet to be determined.

In a new report now published in Nature, Ioannis Manolaridis and colleagues presented high-resolution cryoelectron microscopy structures of human ABCG2 in its substrate-bound state of pre-translocation and in the ATP-bound post-translocation state. The scientists used a mutant protein containing a glutamine in place of the catalytic glutamate (ABCG2EQ) to observe both states. The mutant ABCG2EQ showed reduced ATPase and transport rates to enable conformational trapping suited for the structural studies. In the substrate-bound state of the study, about halfway across the membrane in a cytoplasm-facing cavity, a single molecule of estrone-3-sulfate (E1S) was integrated for functional studies.

Shifts - Transmembrane - Domain - Result - ATP-induced

Rigid-body shifts of the transmembrane domain were observed as a result of ATP-induced conformational changes, pivoting the nucleotide-binding domains (NBDs) and changes in the relative NBD subdomain orientation. In its mechanism of action, ABCG2 harnessed the energy of ATP binding to extrude E1S and other endogenous substrates. Based on the size and binding affinity of compounds it was therefore possible to distinguish substrates from inhibitors in the study.

The authors first established that replacing the catalytic glutamate E211 with a glutamine in the Walker B motif (phosphate binding sequence) would result in mutants...
(Excerpt) Read more at: phys.org
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