Structure and mechanism of Zn^(2+)- transporting P-type ATPases

Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis. In prokaryotes and photosynthetic eukaryotes, Zn2+-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn2+ and related elements. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2·P_i) of ZntA from Shigella sonnei, determined at 3.2 Å and 2.7 Å resolution, respectively. The structures reveal a similar fold to Cu^+-ATPases, with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn^(2+) ions by the transporter. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including C392, C394 and D714. The pathway closes in the E2·P_i state, in which D714 interacts with the conserved residue K693, which possibly stimulates Zn^(2+) release as a built-in counter ion, as has been proposed for H^+-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter transport. These findings suggest a mechanistic link between P_(IB)-type Zn^(2+)-ATPases and P_(III)-type H^+-ATPases and at the same time show structural features of the extracellular release pathway that resemble P_(II)-type ATPases such as the sarcoplasmic/endoplasmic reticulum Ca^(2+)-ATPase (SERCA) and Na^+, K^+-ATPase. These findings considerably increase our understanding of zinc transport in cells and represent new possibilities for biotechnology and biomedicine.