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Crystal structure of the vitamin B3 transporter PnuC, a full-length SWEET homolog

Nature Structural & Molecular Biology volume 21, pages 1013–1015 (2014)Cite this article

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Abstract

PnuC transporters catalyze cellular uptake of the NAD+ precursor nicotinamide riboside (NR) and belong to a large superfamily that includes the SWEET sugar transporters. We present a crystal structure of Neisseria mucosa PnuC, which adopts a highly symmetrical fold with 3 + 1 + 3 membrane topology not previously observed in any protein. The high symmetry of PnuC with a single NR bound in the center suggests a simple alternating-access translocation mechanism.

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Figure 1: Structure of PnuC.
Figure 2: Symmetry of the six-helix core of PnuC.
Figure 3: Nicotinamide riboside (NR) binding to PnuC.

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References

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Acknowledgements

We thank K. Luck for help with protein production and A.J.M. Driessen for use of his ITC machine. The research leading to these results received funding from the European Community's Seventh Framework Programme (FP7/2007–2013) under BioStruct-X (to D.J.S. and A.G., grant agreement 283570). This work was funded by the Netherlands Organisation for Scientific Research (NWO) (NWO ECHO grant 711.011.001 and NWO Vici grant 865.11.001 to D.J.S.) and the European Research Council (ERC) (ERC Starting Grant 282083 to D.J.S.). The European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS) are acknowledged for beamline facilities.

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Author notes

  1. Michael Jaehme and Albert Guskov: These authors contributed equally to this work.

Authors and Affiliations

  1. University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Groningen, the Netherlands

    Michael Jaehme, Albert Guskov & Dirk Jan Slotboom

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  1. Michael Jaehme
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  2. Albert Guskov
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  3. Dirk Jan Slotboom
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All authors designed experiments, performed experiments, analyzed data and wrote the manuscript.

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Correspondence to Dirk Jan Slotboom.

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Integrated supplementary information

Supplementary Figure 1 The chemical structures of nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) and nicotinamide adenine dinucleotide (NAD).

Supplementary Figure 2 Alignment of PnuC proteins.

The residues are colored according to their conservation (the darker the color the better the conservation). The positions of the TMs are shown as colored bars above the sequences, with the color-coding as in Figure 1. Residues involved in substrate binding are indicated by asterisks, and gate residues discussed in the text are indicated by the hash signs. The hydrophilic ladder on TM5 is indicated by a box. The numbers refer to the PnuC sequence from N. mucosa.

Supplementary Figure 3 Structural details of PnuC.

(a) Experimental anomalous map (countered at 6σ), indicating positions of 24 Selenium atoms. The backbone of the PnuC trimer is shown in gray.

(b) 2Fo-Fc electron density map (contoured at 2σ) for PnuC. Only one protomer is shown for clarity.

(c) Tryptophan residues and detergent molecules. The approximate position of the membrane boundaries (Fig. 1b thickness ∼27 Å) was defined by the Trp residues (shown as magenta spheres excluding Trp182 and Trp185, which are located in the binding pocket) and bound detergent molecules (n-octyl-ß-D-glucoside, cyan and red spheres). Tryptophans are preferably found in the headgroup regions of lipid bilayers.

Supplementary Figure 4 Static light scattering (SEC-MALLS) analysis of PnuC from E. coli (24 kDa, top) and Paenibacillus sp. (25 kDa, bottom).

The chromatogram from a size exclusion experiment (black line) and the calculated molar masses of the protein (red), the detergent micelle (DDM, green) and the protein-detergent micelle (blue) are shown. The experiment shows that both proteins are monomeric in detergent solution.

Supplementary Figure 5 The substrate-binding site of PnuC.

(a) Stereo image of 2Fo-Fc electron density map (contoured at 1.5σ) for the bound NR molecule and side chains of amino acids forming the binding site.

(b) Sliced-through surface representation of PnuC showing TM3 (green), TM 7 (red) and the NR molecule (yellow). On the periplasmic side two symmetry-related tyrosines (Tyr95 and Tyr217) are located at the same height as the 5'OH group of the ribose, but do not close the binding pocket. Instead, a cavity extends for one more turn of the TMs.

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Supplementary Figures 1–5 and Supplementary Table 1 (PDF 17577 kb)

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Jaehme, M., Guskov, A. & Slotboom, D. Crystal structure of the vitamin B3 transporter PnuC, a full-length SWEET homolog. Nat Struct Mol Biol 21, 1013–1015 (2014). https://doi.org/10.1038/nsmb.2909

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