Abstract
Cardiolipin (CL) was shown to bound to the dimer interface of NhaA Na+/H+ antiporter. Here, we explore the cardiolipin-NhaA interaction both in vitro and in vivo. Using a novel and straightforward in-vitro assay in which n-dodecyl β-D maltoside (DDM) detergent is used to delipidate the dimer interface and to split the dimers into monomers; the monomers are subsequently exposed to cardiolipin or the other E. coli phospholipids. Most efficient reconstitution of dimers is observed by cardiolipin. This assay is likely to be applicable to future studies of protein–lipid interactions. In-vivo experiments further reveal that cardiolipin is necessary for NhaA survival. Although less efficient phosphatidyl-glycerol (PG) can also reconstitute NhaA monomers to dimers. We also identify a putative cardiolipin binding site. Our observations may contribute to drug design, as human NhaA homologues, which are involved in severe pathologies, might also require specific phospholipids.
Highlights
Na+/H+ antiporters are found in the membranes of most eukaryotic and prokaryotic cells and are essential for homeostasis of intracellular pH, sodium ion concentration, and volume[1]
Intermolecular cross-linking studies have shown that dodecyl β-D maltoside (DDM) may affect the integrity of the NhaA interface in particular[22] suggesting that interfacial lipids may be important for NhaA dimerization, such that exposure to DDM may delipidate the interface and split NhaA dimers into monomers
Pre-incubation at 3% DDM yielded 85% monomers and 15% dimers, whereas pre-incubation at 5% DDM yielded 95% monomers (Fig. 3 top panel, first lane). These observations strongly suggest that, as expected, DDM delipidates the NhaA dimers, and that this delipidation extracts lipids that are needed for maintaining the dimers
Summary
Na+/H+ antiporters are found in the membranes of most eukaryotic and prokaryotic cells and are essential for homeostasis of intracellular pH, sodium ion concentration, and volume[1]. The NhaA monomer comprises 12 transmembrane (TM) helices, packed in two domains: an interface domain, which connects two monomers of NhaA into a dimer (TMs I, II, VI–IX) (Fig. 1); and a core domain, which is involved in function (TMs III–V and X–XII). This structure represents a unique fold called the NhaA structural fold[7]. Lipids were suggested to be essential for the dimerization of two dimeric secondary transporters: NhaA17 and LeuT19, suspension (12 μL, 1.7 μg protein, 7.6 μM NhaA) were prepared.
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