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Abstract

Na⁺/H⁺ antiporters are pH-dependent membrane transport proteins that maintain the homeostasis of H⁺ and Na⁺ in living cells. MjNhaP1 from Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85°C, was cloned and expressed in Escherichia coli. Two-dimensional crystals were obtained from purified protein at pH 4. Electron cryomicroscopy yielded an 8 Å projection map. Like the related E. coli antiporter NhaA, MjNhaP1 is a dimer, but otherwise the structures of the two antiporters differ significantly. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4–6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1 was studied in situ. A major change in density distribution within the helix bundle, and a ∼2 Å shift in the position of the helix bundle relative to the dimer core occurred at pH 6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter.