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Crystal structure of a SLC11 (NRAMP) transporter reveals the basis for transition-metal ion transport

Nature Structural & Molecular Biology volume 21, pages 990–996 (2014)Cite this article

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Abstract

Members of the SLC11 (NRAMP) family transport iron and other transition-metal ions across cellular membranes. These membrane proteins are present in all kingdoms of life with a high degree of sequence conservation. To gain insight into the determinants of ion selectivity, we have determined the crystal structure of Staphylococcus capitis DMT (ScaDMT), a close prokaryotic homolog of the family. ScaDMT shows a familiar architecture that was previously identified in the amino acid permease LeuT. The protein adopts an inward-facing conformation with a substrate-binding site located in the center of the transporter. This site is composed of conserved residues, which coordinate Mn2+, Fe2+ and Cd2+ but not Ca2+. Mutations of interacting residues affect ion binding and transport in both ScaDMT and human DMT1. Our study thus reveals a conserved mechanism for transition-metal ion selectivity within the SLC11 family.

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Figure 1: Transition-metal ion binding and transport.
Figure 2: ScaDMT structure.
Figure 3: Ion coordination.
Figure 4: Transition-metal ion selectivity.
Figure 5: Cd2+ binding to ScaDMT binding-site mutants.
Figure 6: Functional properties of DMT1 binding-site mutants.
Figure 7: Transport mechanism.

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Acknowledgements

This research was supported by the Swiss National Science Foundation through the National Centre of Competence in Research TransCure. We thank the staff of the X06SA beamline for support during data collection, B. Blattman and C. Stutz-Ducommun of the Protein Crystallization Center at the University of Zurich for support with crystallization, B. Dreier for help with MALS experiments, the Center for Microscopy and Image Analysis at the University of Zurich for help with freeze-fracture EM, M. Hediger (University of Bern) for providing the cDNA of human DMT1 and E. Beke (Vrije Universiteit Brussel) for help with nanobody selection. All members of the Dutzler laboratory are acknowledged for help in all stages of the project. E.R.G. acknowledges a long-term postdoctoral fellowship from the Human Frontier Science Program (LT-00899/2008). I.A.E. is affiliated with the Biomolecular Structure and Mechanism PhD program of the University of Zurich (UZH) and the Swiss Federal Institute of Technology (ETH) Zurich. Data collection was performed at the X06SA beamline at the Swiss Light Source of the Paul Scherrer Institute.

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Author notes

  1. Eric R Geertsma

    Present address: Present address: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Frankfurt am Main, Germany.,

Authors and Affiliations

  1. Department of Biochemistry, University of Zurich, Zurich, Switzerland

    Ines A Ehrnstorfer, Eric R Geertsma & Raimund Dutzler

  2. Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium

    Els Pardon & Jan Steyaert

  3. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Els Pardon & Jan Steyaert

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Contributions

I.A.E. carried out all experiments except for the initial nanobody selection. E.R.G. supported the high-throughput expression screening and transport assays and initiated nanobody selection by phage display. E.P. performed immunization, cloned and expressed nanobodies and performed the initial selections. J.S. supervised nanobody production. R.D. assisted I.A.E. in structure determination. I.A.E. and R.D. jointly planned the experiments, analyzed the data and wrote the manuscript.

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Correspondence to Raimund Dutzler.

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Integrated supplementary information

Supplementary Figure 1 Sequence alignment.

Sequences of ScaDMT and human DMT1 (isoform 3) were aligned with ClustalW. Identical residues are highlighted in green and homologous residues in yellow. Numbering corresponds to ScaDMT. Secondary structure elements are shown below. Labels indicate: (black triangle) Start of the truncated construct ScaDMTtru, (red circle) residues of the transition metal binding site, (blue circle) residues involved in pH dependence and H+ transport of DMT1, (green circle) DMT1 disease mutation in the Belgrade rat and the mk/mk mouse and (brown circle) DMT1 disease mutations in human.

Supplementary Figure 2 Multiangle light-scattering and freeze-fracture EM.

Gel filtration and light scattering results for different protein constructs in the detergent DM. Continuous black traces correspond to the UV280 elution profiles. The respective molecular weight of the protein-detergent complex obtained from light scattering is shown at its corresponding position on the chromatogram in green, the molecular weight of the protein component alone in red. Panels show (a) ScaDMT, (b) ScaDMTtru, (c) nanobody, (d) ScaDMT–nanobody complex, (e) ScaDMTtru–nanobody complex. The first peak in panels (c) and (d) corresponds to a homo-dimer of the respective transporter-nanobody complex. (f) Freeze-fracture electron micrograph of proteoliposomes containing ScaDMT. The transporter was reconstituted at a protein to lipid ratio of 1:40 (w/w).

Supplementary Figure 3 Electron density.

(a) Stereo view of the ion-binding region. Experimental electron density calculated at 3.5 Å with Se-Met SAD phases that were improved by solvent flattening and cyclic 2-fold NCS symmetry averaging (blue mesh, contoured at 1 σ) is superimposed on the refined model of the ScaDMTtru–NB complex. (b) The same region of the protein is shown with 2Fo-Fc electron density superimposed (cyan mesh, contoured at 1 σ). The density at 3.1 Å was calculated with phases from the refined model. (c) Anomalous difference electron density of Se atoms (calculated at 3.5 Å and contoured at 5 σ) superimposed on the ScaDMT structure. Protein is shown as Cα-trace, methionine side-chains as sticks. (d) Anomalous difference density of data collected at a wavelength of 1.95 Å. Electron density calculated at 4.5 Å and contoured at 4 σ is superimposed on the model. The density shows peaks for sulfur atoms of methionine and cysteine residues but not for bound Ca2+. (e) Anomalous difference density of Cs+ (calculated at 4.5 Å and contoured at 5 σ, red) is shown superimposed on the ScaDMT structure. ScaDMT is represented as Cα-trace, the position of Mn2+ as black sphere.

Supplementary Figure 4 Nanobody complex and comparison with related proteins.

Stereo views of ScaDMT–nanobody interactions. (a) Structure of the ScaDMT–nanobody complex. Proteins are displayed as Cα-trace. The coloring of the protein is as in Fig. 2, the nanobody is colored in green. (b) Close-up of the interaction interface. Interacting residues are shown as sticks. (c) Stereo view of the superposition of ScaDMT on the inward-facing conformation of LeuT. The superposition was based on the Cα positions of equivalent residues in transmembrane helices (RMSD 3.2 Å). ScaDMT is colored in orange, LeuT in red. (d) Stereo view of the superposition of ScaDMT on the inward-facing conformation of vSGLT. The superposition was based on the Cα positions of equivalent residues in transmembrane helices (r.m.s.d. 3.9 Å). ScaDMT is colored in orange, vSGLT in green.

Supplementary Figure 5 Structure of full-length ScaDMT.

Stereo view of the full-length ScaDMT structure at 6.5 Å resolution. The structure shows one of the two transporters present in the asymmetric unit. The electron density calculated from phases obtained by molecular replacement with the program Phaser is colored in blue, electron density after rigid-body refinement in PHENIX is shown in cyan. Both electron densities are contoured at 1 σ. ScaDMT is colored in orange, α-helix 1a of LeuT from the superimposed model is shown in red. (a) View from within the membrane, (b) view from the cytoplasm.

Supplementary Figure 6 Transport properties of ScaDMT binding-site mutants.

Transport of Mn2+ into proteoliposomes containing the ScaDMT binding site mutants D49A (a), N52A (b) and M226A (c). Left: Time-dependent quenching of the fluorophore calcein, that is trapped inside the vesicle, upon addition of 300 μM Mn2+ to the external medium. Addition of Mn2+ and the ionophore calcimycin (Cal.), which acts as Mn2+/H+ exchanger, are indicated. A control trace from liposomes devoid of protein upon addition of 300 μM Mn2+ is shown in black, transport into proteoliposomes containing WT is shown in grey. Compared to WT, the proteoliposomes containing the mutants D49A, N52A and M226A show decreased activity. Right: ScaDMT-mediated transport of Mn2+ in the presence of Cd2+. Time-dependent quenching of the fluorophore calcein, was monitored upon addition of 100 μM of Mn2+ and 100 μM Cd2+ to the external medium. Transport by WT under the same conditions is shown in grey for comparison, traces from liposomes devoid of protein are shown in black. In all cases the presence of Cd2+ decreases the transport of Mn2+ into liposomes.

Supplementary Figure 7 Location of disease mutations.

The position of disease causing mutations in DMT1 in rodents (green) and human (magenta) are mapped on equivalent positions on the ScaDMT structure. The numbering corresponds to human DMT1. The protein is shown as Cα-trace, the mutated residues are indicated as spheres, the approximate membrane boundaries are indicated in grey.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 5088 kb)

Supplementary Data Set 1

Western blot of human DMT1 mutants (PDF 178 kb)

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Ehrnstorfer, I., Geertsma, E., Pardon, E. et al. Crystal structure of a SLC11 (NRAMP) transporter reveals the basis for transition-metal ion transport. Nat Struct Mol Biol 21, 990–996 (2014). https://doi.org/10.1038/nsmb.2904

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