Probing native metal ion association sites through quenching of fluorophores in the nucleotide-binding domains of the ABC transporter MsbA.
Biochem J
; 474(12): 1993-2007, 2017 05 30.
Article
in En
| MEDLINE
| ID: mdl-28432259
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Bacterial Proteins
/
Models, Molecular
/
Adenosine Triphosphate
/
ATP-Binding Cassette Transporters
/
Escherichia coli Proteins
/
Nickel
Type of study:
Prognostic_studies
/
Risk_factors_studies
Language:
En
Journal:
Biochem J
Year:
2017
Document type:
Article
Affiliation country: