Abstract
We examined substrate-induced conformational changes in MjNhaP1, an archaeal electroneutral Na(+)/H(+)-antiporter resembling the human antiporter NHE1, by electron crystallography of 2D crystals in a range of physiological pH and Na(+) conditions. In the absence of sodium, changes in pH had no major effect. By contrast, changes in Na(+) concentration caused a marked conformational change that was largely pH-independent. Crystallographically determined, apparent dissociation constants indicated ∼10-fold stronger Na(+) binding at pH 8 than at pH 4, consistent with substrate competition for a common ion-binding site. Projection difference maps indicated helix movements by about 2 Å in the 6-helix bundle region of MjNhaP1 that is thought to contain the ion translocation site. We propose that these movements convert the antiporter from the proton-bound, outward-open state to the Na(+)-bound, inward-open state. Oscillation between the two states would result in rapid Na(+)/H(+) antiport. DOI: http://dx.doi.org/10.7554/eLife.01412.001.
Highlights
Na+/H+ antiporters are ubiquitous and essential secondary-active transporters found in the cell membranes of all organisms
Na+/H+ antiporters belong to the superfamily of cation/proton antiporters (CPA), which include the CPA1 and CPA2 subfamilies as main branches (Brett et al, 2005)
In the CPA1 antiporters, one of the aspartates is replaced by an asparagine, which likewise is thought to participate in ion-binding and translocation (Hellmer et al, 2003; Goswami et al, 2011)
Summary
Na+/H+ antiporters are ubiquitous and essential secondary-active transporters found in the cell membranes of all organisms. They play crucial roles in the regulation of intracellular pH, sodium homeostasis, and cell volume. The best-known member of the CPA2 subfamily is the Na+/H+ antiporter NhaA from E. coli. EcNhaA enables E. coli to survive at high salinity or alkaline pH (Padan and Schuldiner, 1994), making use of the proton-motive force to extrude sodium from the cell (Figure 1). Other members of the CPA2 subfamily include the plant CHX transporters and the mammalian NHA transporters. Wellknown representatives of the CPA1 subfamily include the medically important mammalian NHE exchangers and the archaeal NhaP antiporters (Brett et al, 2005)
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