1 Department of Molecular Biology and Genetics - DANDRITE, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University2 DANDRITE - Nissen Group, DANDRITE, Interfaculty, Aarhus University3 Department of Molecular Biology and Genetics - Structural Biology, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University4 Interdisciplinary Nanoscience Center - INANO-MBG, Gustav Wied 10, Interdisciplinary Nanoscience Center, Science and Technology, Aarhus University5 Science for Life Laboratory, Theoretical and Computational Biophysics, Department of Theoretical Physics, Swedish e-Science Research Center, KTH Royal Institute of Technology6 Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA.7 Department of Molecular Biology and Genetics - DANDRITE, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University8 Department of Molecular Biology and Genetics - Structural Biology, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University
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 catalysis1. 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 elements2, 3. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2·Pi) 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 Zn2+ 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·Pi state, in which D714 interacts with the conserved residue K693, which possibly stimulates Zn2+ 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 PIB-type Zn2+-ATPases and PIII-type H+-ATPases and at the same time show structural features of the extracellular release pathway that resemble PII-type ATPases such as the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase4, 5 (SERCA) and Na+, K+-ATPase6. These findings considerably increase our understanding of zinc transport in cells and represent new possibilities for biotechnology and biomedicine.