Nucleotides and transported substrates modulate different steps of the ATPase catalytic cycle of MRP1 multidrug transporter

András Kern, Zsófia Szentpétery, Károly Liliom, Éva Bakos, Balázs Sarkadi, András Váradi

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Abstract

The human ABC (ATP-binding cassette) transporter MRP1 (human multidrug-resistance-associated protein 1; ABCC1) is involved in the cellular extrusion of conjugated metabolites and causes multidrug resistance in tumour cells. The transport of substrate molecules by ABC proteins is energized by ATP hydrolysis, performed by two co-operating ABC units. Orthovanadate (Vi), a non-covalent inhibitor of the ABC ATPases, was found to catalyse a photo-oxidative cleavage of various ATP-binding proteins. In the present study, we have identified three Vi-cleavage sites within MRP1, and found that the cleavage reactions were variably modulated by the presence of nucleotides and by transported substrates. We concluded that Vi cleavage of MRP1 at Site I detects conformational changes due to the binding of MgATP. In contrast, Site II could be identified as part of the substrate-modulated catalytic cycle, probably containing an MRP1·MgADP·Vi transition-state-like complex. Cleavage at Site III was modulated by both the binding and hydrolysis of MgATP, in a biphasic pattern, which was also affected by the presence of transported substrates. We detected two different allosteric effects and found that they control two consecutive steps of the MRP1 ATPase catalytic cycle. Nucleotide binding to the low-affinity site accelerated the formation of the pre-hydrolytic intermediate in the other catalytic centre. Interaction of the transporter with its transported substrates stimulated a later reaction of the hydrolytic cycle, the formation of the post-hydrolytic intermediate, which could be detected in both catalytic sites by the experimental strategy used.

Original languageEnglish
Pages (from-to)549-560
Number of pages12
JournalBiochemical Journal
Volume380
Issue number2
DOIs
Publication statusPublished - Jun 1 2004

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Keywords

  • ATP-binding cassette transporter (ABC transporter)
  • ATPase catalytic cycle
  • Multidrug-resistance-associated protein 1 (MRP1)
  • Pre- and post-hydrolytic intermediate
  • Transition state
  • Vanadate cleavage

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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