Boosted coupling of ATP hydrolysis to substrate transport upon cooperative estradiol-17-β-D-glucuronide binding in a Drosophila ATP binding cassette type-C transporter

Agnes Karasik, Kaitlyn Victoria Ledwitch, Tamás Arányi, András Váradi, Arthur Roberts, Flóra Szeri

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

ATP binding cassette type-C (ABCC) transporters move molecules across cell membranes upon hydrolysis of ATP; however, their coupling of ATP hydrolysis to substrate transport remains elusive. Drosophila multi-drug resistance-associated protein (DMRP) is the functional ortholog of human long ABCC transporters, with similar substrate and inhibitor specificity, but higher activity. Exploiting its high activity, we kinetically dissected the catalytic mechanism of DMRP byusing E2-D-glucuronide (E2G), the physiologic substrate of human ABCC. We examined the DMRP-mediated interdependence of ATP and E2G in biochemical assays. We observed E2G-dependent ATPase activity to be biphasic at subsaturating ATP concentrations, which implies at least 2 E2G binding sites on DMRP. Furthermore, transport measurements indicated strong nonreciprocal cooperativity between ATP and E2G. In addition to confirming these findings, our kinetic modeling with the Complex Pathway Simulator indicated a 10-fold decrease in the E2G-mediated activation of ATP hydrolysis upon saturation of the second E2G binding site. Surprisingly, the binding of the second E2G allowed for substrate transport with a constant rate, which tightly coupled ATP hydrolysis to transport. In summary, we show that the second E2G binding-similar to human ABCC2-allosterically stimulates transport activity of DMRP. Our data suggest that this is achieved by a significant increase in the coupling of ATP hydrolysis to transport.-Karasik, A., Ledwitch, K. V., Arányi, T., Váradi, A., Roberts, A., Szeri, F. Boosted coupling of ATP hydrolysis to substrate transport upon cooperative estradiol-17-b-D-glucuronide binding in a Drosophila ATP binding cassette type-C transporter. FASEB J. 32, 669-680 (2018). www.fasebj.org.

Original languageEnglish
Pages (from-to)669-680
Number of pages12
JournalFASEB Journal
Volume32
Issue number2
DOIs
Publication statusPublished - Feb 2018

Keywords

  • Allosteric cooperativity
  • COPASI
  • Drosophila MRP
  • EG transport
  • Kinetic modeling

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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