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Structure and mechanism of Zn2+-transporting P-type ATPases

Nature volume 514, pages 518–522 (2014)Cite this article

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Abstract

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.

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Figure 1: Structures of the S. sonnei Zn2+-ATPase.
Figure 2: Functional studies of zinc, cadmium, lead and counter-ion transport in S. sonnei ZntA.
Figure 3: Details of the S. sonnei ZntA structures.
Figure 4: Putative zinc transport mechanism of ZntA.

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the S. sonnei ZntA (UniProt ID, Q3YW59) E2–AlF4− and E2–BeF3− crystal structures have been deposited in the Protein Data Bank (PDB) under accession numbers 4UMW and 4UMV.

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Acknowledgements

We thank J. L. Karlsen for support with crystallographic computing. O.S. and H.E.A. are supported by the Graduate School of Science and Technology at Aarhus University. G.M. is supported by a Marie Curie International Outgoing Fellowship (European Commission, grant no. 252961). M.A. was supported by a Marie Curie Career Integration Grant (FP7-MC-CIG-618558). P.N. was supported by an advanced research grant from the European Research Council (250322 Biomemos), and P.G. was supported, in the later stage, by the Lundbeck Foundation and the Swedish Research Council (K2013-99X-22251-01-5). We are grateful for assistance with crystal screening from Maxlab, beam lines 911-2/3, and with data collection from the Swiss Light Source, beam line X06SA. Access to synchrotron sources was supported by the Danscatt program of the Danish Council of Independent Research and by BioStruct-X contract 860.

Author information

Author notes

  1. Kaituo Wang & Pontus Gourdon

    Present address: Present addresses: Department of Biomedical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark (K.W. and P.G.); Department of Experimental Medical Science, Lund University, Sölvegatan 19, SE-221 84 Lund, Sweden (P.G.).,

  2. Kaituo Wang and Oleg Sitsel: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Centre for Membrane Pumps in Cells and Disease (PUMPkin), Danish National Research Foundation, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark,

    Kaituo Wang, Oleg Sitsel, Gabriele Meloni, Henriette Elisabeth Autzen, Tetyana Klymchuk, Anna Marie Nielsen, Poul Nissen & Pontus Gourdon

  2. Department of Theoretical Physics, Science for Life Laboratory, Swedish e-Science Research Center, KTH Royal Institute of Technology, SE-171 21 Solna, Sweden,

    Magnus Andersson

  3. Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA,

    Douglas C. Rees

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Contributions

K.W., O.S., T.K. and A.M.N. cloned the S. sonnei ZntA constructs. K.W. and O.S. performed protein purification, crystallization and activity measurements in solution. G.M. conducted the vesicle and zinc binding studies, which were developed with D.C.R. K.W. processed the data and solved the crystal structures, and all authors analysed the results. H.E.A. and M.A. conducted molecular dynamics simulations in the absence and presence of zinc, respectively. P.N. and P.G. designed the project. K.W., O.S. and G.M. generated the figures. P.N. and P.G. wrote the paper, and all authors commented on the paper.

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Correspondence to Pontus Gourdon.

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Extended data figures and tables

Extended Data Figure 1 Topology and reaction cycle of P-type ATPases.

a, Topology of ZntA, CopA and SERCA. Key residues in the HMBD and A, P, N and M domains are highlighted. In ZntA, the negatively charged ion entry funnel and release pathway are outlined. D436 in S. sonnei ZntA is the autophosphorylated/dephosphorylated catalytic aspartate in the DKTGTXT motif of the P domain. C392 and C394 in M4, K693 in M5 and D714 in M6 of S. sonnei ZntA have been proposed to bind zinc in biochemical studies10,15,50. b, The Post–Albers (E1 to E2) reaction cycle of Zn2+-transporting P-type ATPase9,51. Phosphorylation events in the intracellular domains drive large conformational changes that permit alternating access to transport sites in the membrane about 50 Å from the ATP-targeted catalytic aspartate. According to the model, a high-affinity state (E1), which is open to the intracellular space, binds to Zn2+ and enters an occluded state. This state then undergoes phosphorylation. Completion of this event (E1P) triggers the release of the Zn2+, establishing an outward-facing, low-affinity state (E2P). Release of the inorganic phosphate (Pi) yields the fully dephosphorylated conformation (E2), which is followed by restoration of the inward-facing conformation (E1), which initiates a new reaction cycle.

Extended Data Figure 2 Structure-based sequence alignment of S. sonnei ZntA and L. pneumophila CopA.

Helix positions are indicated for S. sonnei ZntA, and noteworthy residues are highlighted. Four of seven amino acid positions in which ZntA differs between S. sonnei and Escherichia coli and that are likely to be functionally irrelevant are highlighted in grey. The high-affinity ion-binding residues C392, C394 and D714 are indicated in purple; the catalytically phosphorylated aspartate and the dephosphorylating TGE motif are highlighted in green. E202 and K693, which are possibly involved in ion release, are marked in black. The alignment was performed using SALIGN52.

Extended Data Figure 3 Electron density of the determined E2·Pi state of S. sonnei ZntA.

a, Final 2Fo − Fc electron density of S. sonnei ZntA in the E2·Pi state. The density is contoured at 1σ, and the view is equivalent to that shown in Fig. 1a. b, c, Se-Met peaks calculated using Se-SAD (Se single-wavelength anomalous diffraction) data and phases obtained from molecular-replacement-guided model building. The anomalous difference Fourier map is contoured at 3σ. A view of the entire protein (b), and a view of the M domain (c) are shown.

Extended Data Figure 4 Functional assays of S. sonnei ZntA.

a, Wild-type and ΔHMBD (inset) S. sonnei ZntA ATPase activity is stimulated by Zn2+, Cd2+ and Pb2+. ATPase activity (normalized; the activity in the presence of Zn2+ is set at 100% for the wild type and ΔHMBD, respectively) was determined using the Baginski assay (see Methods for details). This ion stimulation profile matches the one observed for ZntA from E. coli53. The mean + s.d. of technical replicates is shown (n = 3). b, Effect of K+, Na+ and Mg2+ on S. sonnei ZntA activity. The ATPase activity of wild-type S. sonnei ZntA in detergent micelles or upon reconstitution in proteoliposomes, in buffers containing exclusively Na+ or K+, as determined by the Baginski assay, is shown. For Mg2+, the activity was in the proteoliposomes for internal buffers with or without MgCl2. The mean + s.d. of technical replicates is shown (n = 3). c, Zn2+ and H+ transport across vesicle membranes. Zn2+ transport of wild-type and D436N S. sonnei ZntA proteoliposomes monitored using the zinc-selective chelator FluoZin-1 (left). H+ counter-ion transport in wild-type S. sonnei ZntA or Ca2+-ATPase LMCA proteoliposomes monitored using the pH indicator pyranine (right).

Extended Data Figure 5 Structural comparison of ZntA and CopA.

a, Difference between the extracellular loops of S. sonnei ZntA and L. pneumophila CopA. S. sonnei ZntA is coloured as in Fig. 1a and L. pneumophila CopA is in dark green, and the proteins have been aligned on helices M5 and M6. Note that the loops are substantially longer in L. pneumophila CopA than in S. sonnei ZntA, which is a conserved difference between Cu+- and Zn2+-transporting P-type ATPases (see also b). b, c, Comparison of the extracellular loop lengths of ZntA (b) and CopA (c). The lengths of the loops in S. sonnei ZntA and L. pneumophila CopA are shown, as well as averages based on 521 ZntA-type proteins and 617 CopA-type proteins (with less than 99% and 95% sequence identity within the ZntA and CopA sequences, respectively).

Extended Data Figure 6 The phosphorylation site of S. sonnei ZntA.

The domains are coloured as in Fig. 1a. AlF4−/BeF3− (Al in orange, Be in green and F in cyan) and the Mg2+ ion (grey) are associated with D436 (in the DKTGTXT motif of the P domain) at the interface between the A and P domains. D436, T438, T583, D628, N631 and D632 (in the P domain), as well as T288, G289 and E290 (the TGE motif in the P domain that is associated with dephosphorylation), are shown as sticks. Water molecules are shown as red spheres (not modelled for the E2P state). a, The E2P–BeF3−-bound state. The catalytic D436 is protected from the TGE loop. b, The E2·Pi–AlF4−-bound state. E290 of the TGE loop probably activates a water molecule for dephosphorylation as observed in the equivalent E2·Pi state of SERCA1A and CopA4,5,11.

Extended Data Figure 7 The extracellular pathway.

a, The extracellular fraction of the E2–AlF4− crystal structure. Functionally important residues are shown as sticks, and the protein is coloured as in Fig. 1a. The final 2Fo − Fc electron density is contoured at 1σ. The view is equivalent to the one in Fig. 3d. b, Dynamics of E202 in a 60-ns molecular dynamics simulation of the E2–BeF3− structure in a dioleoylphosphatidylcholine (DOPC) membrane in the absence of zinc. Selected residues are shown as sticks. Representative E202 conformations were captured at 16, 25 and 30 ns from snapshots aligned according to backbone Cαs of M1–M4. The orientation of E202 at 16 ns resembles how this side chain appears in the E2–AlF4− state, while the flexibility observed throughout the simulation agrees with the observed poor electron density of the side chain in the E2–BeF3− state (see Fig. 3b). Note that there are two distorted lipids at the release pathway that may assist in Zn2+ release (vdW spheres represent lipid phosphates). c, Distance between the centre of mass of the Cδ of the E202 side chain and the NZ of the K693 side chain during the 60-ns simulations of the E2–AlF4− and E2–BeF3− S. sonnei ZntA structures in the absence of zinc, as a running average over five consecutive frames of each trajectory. d, The release pathway and accompanying protein interactions experienced by Zn2+ in a steered molecular dynamics simulation originating from the centre of mass of residues C392, C394 and D714. The transmembrane domain, lipid phosphates and water within 7 Å of the protein are coloured as in b. e, The number of Zn2+–protein interactions with a 5 Å cut-off during steered molecular dynamics (SMD) simulations. Error bars correspond to counts from ten independent simulations with pulling speeds on Zn2+ of 10–20 Å ns−1.

Extended Data Figure 8 Surface charge distribution and docking of the HMBD to S. sonnei ZntA.

a, Four views of the overall structure of E2–AlF4−. The view to the left is equivalent to that in Fig. 1a. The charge distribution complies with the positive-inside rule for membrane proteins54. The putative ion entry funnel is indicated with a black arrow. b–d, Docking of the HMBD to S. sonnei ZntA. The apo-HMBD of E. coli ZntA (PDB ID, 1MWY13) docks to the entry site region of S. sonnei ZntA using electrostatic complementation and van der Waals interactions, as predicted by the ClusPro 2.0 server36 (b). Equivalent view to that in a of S. sonnei ZntA without the HMBD (c). View of the isolated HMBD, rotated 180° relative to a to show the surface complementary to S. sonnei ZntA (d). The ion-binding cysteine residues C15 and C18 are highlighted.

Extended Data Table 1 Data collection, phasing and refinement statistics
Full size table
Extended Data Table 2 Statistical analysis of the ion entry region of S. sonnei ZntA
Full size table

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Wang, K., Sitsel, O., Meloni, G. et al. Structure and mechanism of Zn2+-transporting P-type ATPases. Nature 514, 518–522 (2014). https://doi.org/10.1038/nature13618

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