Probing native metal ion association sites through quenching of fluorophores in the nucleotidebinding domains of the ABC transporter MsbA

Daiki Tatsumi, Kei Nanatani, Yuto Koike, Kiyoto Kamagata, Satoshi Takahashi, Ayumu Konno, Tadaomi Furuta, Minoru Sakurai, Nobuyuki Uozumi

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

ATP-binding cassette (ABC) transporters are ubiquitously present in prokaryotic and eukaryotic cells.Binding of ATP to the nucleotide-binding domains (NBDs) elicits major conformational changes of the transporters resulting in the transport of the substrate across the membrane.The availability of a crystal structure of the NBDs enabled us to elucidate the local structure and small-scale dynamics in the NBDs.Here, we labeled the ABC transporter MsbA, a homodimeric flippase from Escherichia coli, with a fluorescent probe, Alexa532, within the NBDs.ATP application elicited collisional quenching, whereas no quenching was observed after the addition of ATP analogs or ATP hydrolysis inhibitors.The Alexa532-conjugated MsbA variants exhibited transition metal ion Förster resonance energy transfer (tmFRET) after the addition of Ni2+, and ATP decreased this Ni2+-mediated FRET of the NBDs.Structure modeling developed from crystallographic data and examination of tmFRET measurements of MsbA variants in the absence of ATP revealed the presence of metal ion-associated pockets (MiAPs) in the NBDs.Three histidines were predicted to participate in chelating Ni2+ in the two possible MiAPs.Performing histidine-substitution experiments with the NBDs showed that the dissociation constant for Ni2+ of MiAP2 was smaller than that of MiAP1.The structural allocation of the MiAPs was further supported by showing that the addition of Cu2+ resulted in higher quenching than Ni2+.Taken together, the present study showed that the NBDs contain two native binding sites for metal ions and ATP addition affects the Ni2+-binding activity of the MiAPs.

Original languageEnglish
Pages (from-to)1993-2007
Number of pages15
JournalBiochemical Journal
Volume474
Issue number12
DOIs
Publication statusPublished - 2017 Jun 15

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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