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PMID:18344567

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Citation

Eckford, PD and Sharom, FJ (2008) Functional characterization of Escherichia coli MsbA: interaction with nucleotides and substrates. J. Biol. Chem. 283:12840-50

Abstract

The Escherichia coli MsbA protein is a 65-kDa member of the ATP-binding cassette superfamily. It is thought to function as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA with high ATPase activity was isolated and found to be homodimeric in detergent solution. The protein ATPase activity was inhibited by vanadate and showed variable patterns of stimulation and inhibition by lipid A and other compounds. The intrinsic tryptophan fluorescence of the protein was characterized, and dynamic quenching using acrylamide showed that a conformational change took place on binding of lipid A. Fluorescence quenching was used to characterize the interactions of MsbA with nucleotides and various putative substrates, including lipids, lipid-like compounds, and drugs. MsbA had an apparent binding affinity for ATP of approximately 2 mm and also bound nonhydrolyzable ATP analogs and fluorescent ATP derivatives. The putative substrate lipid A interacted with the protein with an affinity of 6.4 microm. Drugs that are known to be substrates for ABC multidrug transporters also interacted with MsbA with affinities in the range 0.25-50 microm. This study represents the first use of fluorescence approaches to estimate MsbA binding affinities for nucleotides and putative transport substrates.

Links

PubMed Online version:10.1074/jbc.M708274200

Keywords

ATP-Binding Cassette Transporters/genetics; ATP-Binding Cassette Transporters/isolation & purification; ATP-Binding Cassette Transporters/metabolism; Acrylamide/pharmacology; Adenosine Triphosphatases/antagonists & inhibitors; Adenosine Triphosphatases/metabolism; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Drug Resistance, Multiple/drug effects; Enzyme Inhibitors/pharmacology; Escherichia coli/drug effects; Escherichia coli/genetics; Escherichia coli/metabolism; Lipid Metabolism; Molecular Structure; Nucleotides/metabolism; Protein Binding; Solutions; Substrate Specificity

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:MSBA

enables

GO:0034040: ATPase-coupled lipid transmembrane transporter activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:MSBA

enables

GO:0008144: drug binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:MSBA

enables

GO:0008289: lipid binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:MSBA

enables

GO:0034040: lipid-transporting ATPase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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