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ATPase activity of the MsbA lipid flippase of Escherichia coli

William T Doerrler1, Christian R H Raetz

  • 1Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

The Journal of Biological Chemistry
|July 18, 2002
PubMed

Insights

Escherichia coli MsbA, an essential inner membrane lipid flippase, functions as a lipid-activated ATPase. Hexaacylated lipid A potently stimulates MsbA activity, suggesting a role in lipopolysaccharide transport.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Escherichia coli MsbA is an essential inner membrane protein involved in lipid transport.
  • MsbA shares homology with mammalian multidrug resistance proteins.
  • Loss of MsbA function leads to the accumulation of lipids in the inner membrane.

Purpose of the Study:

  • To investigate the biochemical activity of purified E. coli MsbA.
  • To determine if MsbA exhibits ATPase activity and if it is regulated by lipids.
  • To characterize the interaction between MsbA and specific lipid molecules.

Main Methods:

  • Overexpression and purification of N-terminally hexahistidine-tagged MsbA using nickel affinity chromatography.
  • Solubilization of MsbA with a nonionic detergent.
  • Reconstitution of purified MsbA into liposomes composed of E. coli phospholipids.
  • Assay of ATPase activity in the presence of varying concentrations of ATP, Mg(2+), and specific lipid A derivatives.

Main Results:

  • Purified MsbA demonstrated ATPase activity, which was stimulated by phospholipids.
  • Reconstituted MsbA exhibited an apparent K(m) of 878 µM and V(max) of 37 nmol/min/mg for ATP hydrolysis.
  • Preincubation with 3-deoxy-d-mannooctulosonic acid (Kdo)(2)-lipid A significantly increased ATPase activity (4-5 fold), with half-maximal stimulation at 21 µM.
  • Kdo(2)-lipid A enhanced V(max) to 154 nmol/min/mg and decreased K(m) to 379 µM.
  • Only hexaacylated lipid A species, not precursors like lipid X or lipid IV(A), stimulated MsbA activity.
  • A temperature-sensitive MsbA mutant (A270T) showed reduced ATPase activity at elevated temperatures.

Conclusions:

  • These findings provide the first in vitro evidence that MsbA is a lipid-activated ATPase.
  • Hexaacylated lipid A is a potent activator of MsbA's ATPase activity.
  • MsbA likely plays a crucial role in the transport of hexaacylated lipid A, a key component of lipopolysaccharide, across the inner membrane.

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