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A saposin-lipoprotein nanoparticle system for membrane proteins

Nature Methods volume 13, pages 345–351 (2016)Cite this article

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Abstract

A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptTSo2 by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state.

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Figure 1: Salipro nanoparticles stabilize membrane proteins.
Figure 2: Analysis of Salipro particles with lipids and the T2 channel.
Figure 3: Single-particle cryo-EM of Salipro-POT.
Figure 4: Cryo-EM structure of Salipro-POT.
Figure 5: Stabilization of soluble and functional HIV-1 spike proteins in Salipro nanoparticles.

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Acknowledgements

This work was supported by the European Molecular Biology Organization (EMBO) (long-term fellowship to J.F.), the Swedish Research Council (grants 2014-5583 and 2013-3922 to P.N., grant 2013-4621 to H.G. and grant 2010-4483 to C.J.), the Knut and Alice Wallenberg Foundation (grant 2014.0112 to P.N.), the Swedish Cancer Society (grant 13 0401 to P.N. and grant 14 0473 to H.G.), the Swedish Childhood Cancer Foundation (grant PR2014-0156 to P.N.), Nanyang Technological University (start-up grant to P.N.), Marie Curie Actions Project FP7-People-ITN-2008 (Virus Entry 235649 to H.G.), Stiftelsen Läkare mot AIDS Forskningsfond (to R.L.), Howard Hughes Medical Institute (Y.C.), the US National Institutes of Health (grants R01GM098672, P50GM082250 and 1S10OD020054 to Y.C.), the Karolinska Institutet Center for Biosciences (to C.J.), the China Scholarship Council (fellowship to L.Z.) and the Molecular Medicine Partnership Unit (MMPU) of the University Clinic Heidelberg (A.F.-P.S. and J.A.G.B.) and the European Molecular Biology Laboratory (A.F.-P.S. and J.A.G.B.). We also acknowledge the Protein Science Facility at Karolinska Institutet for cloning assistance and protein purification. Y.C. is an investigator at Howard Hughes Medical Institute.

Author information

Author notes

  1. Jens Frauenfeld

    Present address: Present address: Salipro Biotech AB, Södertälje, Sweden.,

Authors and Affiliations

  1. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Jens Frauenfeld, Fatma Guettou, Per Moberg, Christian Löw & Pär Nordlund

  2. Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden

    Robin Löving & Henrik Garoff

  3. Department of Biochemistry and Biophysics, Keck Advanced Microscopy Laboratory, University of California San Francisco, San Francisco, California, USA

    Jean-Paul Armache & Yifan Cheng

  4. Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Andreas F-P Sonnen & John A G Briggs

  5. Molecular Medicine Partnership Unit, European Molecular Biology Laboratory–Universitätsklinikum Heidelberg, Heidelberg, Germany

    Andreas F-P Sonnen & John A G Briggs

  6. Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden

    Lin Zhu & Caroline Jegerschöld

  7. School of Technology and Health, Royal Institute of Technology, Novum, Huddinge, Sweden.,

    Lin Zhu & Caroline Jegerschöld

  8. EMBL Hamburg, Hamburg, Germany

    Ali Flayhan & Christian Löw

  9. Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California, USA

    Yifan Cheng

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Contributions

J.F. developed the concept, performed experiments, analyzed data and wrote the manuscript. R.L. developed, performed and analyzed HIV-related experiments. J.-P.A. carried out all cryo-EM experiments, including data acquisition, processing and data interpretation. F.G., P.M., A.F. and C.L. purified membrane proteins. A.F.-P.S. prepared grids, collected negative-stain data and conducted image processing on Salipro-T2. C.J. and L.Z. prepared grids and collected negative-stain data on lipid-only Salipro nanoparticles. J.A.G.B., H.G., Y.C. and P.N. contributed project feedback and comments on the manuscript. All authors contributed to data interpretation and preparation of the manuscript.

Corresponding authors

Correspondence to Jens Frauenfeld or Pär Nordlund.

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Competing interests

Some of the authors have filed patent applications related to this work (J.F.: EP 2 745 834, WO 2014/095576; J.F., R.L. and H.G.: WO 2015/036549). J.F. and R.L. are shareholders of Salipro Biotech AB.

Integrated supplementary information

Supplementary Figure 1 Salipro lipid particles.

(a) Gel filtration analysis of Saposin A after incubation with the indicated detergent solubilised lipid solutions. The generation of Saposin A-lipid complexes was unfavorable using PE and an E. coli total lipid extract. The void volume is marked with an asterisk, the peak for monomeric Saposin with (^). (b) Negative-stain electron microscopy images of lipid-only Salipro particles. The scale bar=50 nm. (c) Gel filtration analysis of Saposin A after incubation with varying amounts of lipids (“Lipids 5”: 5 μg, “Lipids 12.5”: 12.5 μg, “Lipids 25”: 25 μg, “Lipids 50”: 50 μg, “Lipids 100”: 100 μg) from a brain-lipid solution (5 mg/ml brain lipids) in a final reaction volume of 65 μl. Peaks for monomeric Saposin are marked with (^) and for Saposin-lipid complexes with (#). (d) Gel filtration analysis of lipid Salipro particles after a 10 min incubation step at the indicated temperatures. The void volume is marked with an asterisk, the peaks for monomeric Saposin with (^) and Saposin-lipid complexes with (#).

Supplementary Figure 2 Incorporation of the bacterial POT transporter.

(a) Gel filtration analysis of purified Salipro-T2 that has been concentrated, frozen, thawed, diluted and subjected to SEC. The void volume is marked with an asterisk and the peak for Salipro-T2 with (<>). (b) Gel filtration analysis of the incorporation of the tetrameric PepT channel into Salipro nanoparticles. The void volume is marked with an asterisk, the peaks for monomeric Saposin with (^), Saposin-lipid complexes with (#) and Salipro-POT with (<>). Note that the gel filtration buffer is 1xPBS pH 7.4. In the absence of Saposin and lipids, the membrane protein POT completely aggregates under these conditions (green line). (c) Gel filtration analysis of the incorporation of the tetrameric PepT channel into Salipro nanoparticles at various pH conditions, as indicated. The void volume is marked with an asterisk, the peaks for monomeric Saposin with (^), Saposin-lipid complexes with (#) and Salipro-POT with (<>). (d) Thermal unfolding analysis with dye-free differential scanning fluorimetry (using two samples each for POT in detergent (1x PBS, pH 7.4, 0.4% NM) and Salipro-POT (1x PBS pH 7.4). Deviation of the mean is indicated. The bacterial peptide transporter is significantly more stable when embedded in Salipro nanoparticles (Tm 72°C) as compared to detergent micelles (POT in Nonyl-β-D-Maltopyranoside, Tm 43°C). (c) Stoichiometry of Salipro-POT. SDS-PAGE of purified Salipro-POT1 and BSA-standards in concentrations as indicated, suggesting a 1:1 ratio of Saposin / POT. Given a tetrameric POT, this means each Salipro-POT contains four Saposin proteins, as confirmed by the cryo-EM structure.

Supplementary Figure 3 Salipro-POT density displayed in two different isosurface levels.

Left: Top and side view of the 3D reconstruction displayed at two different isosurface levels (high in orange, low in grey mesh). At low isosurface level, the density of the Saposin-lipid scaffold is visible. Right: Side and top view, cut within the plane of the membrane as indicated (dashed line).

Supplementary Figure 4 Solubilization of radioactively labeled HIV-1 VLPs with high–critical micelle concentration (CMC) detergents.

HIV-1 VLPs were solubilised in 1x HNC buffer containing 25 mM Anameg-7 (CMC 19.5 mM) (lane 2), 9 mM HEGA-10 (CMC 7 mM) (lane 3), 14 mM C-HEGA-11 (CMC 11.5 mM) (lane 4), 9 mM MEGA-10 (CMC 6-7 mM) (lane 5), 12 mM n18 Octyl-beta-D-Thiomaltopyranoside (OT) (CMC 9 mM) (lane 6), or 10 mM Tetraethylene Glycol Monooctyl Ether (C8E4) (CMC 10 mM) (lane 7), for 10 min on ice (a) or for 30 min at 37°C (b) and analysed by BN-PAGE. VLPs solubilised in TX100 (TX) on ice were analyzed as control (lane 1). Migration of spikes and gp monomers are indicated. Note the dissociation of spikes into monomers by the 37 °C incubation.

Supplementary Figure 5 Schematic illustration of the Salipro–HIV spike purification.

For detailed description see material and methods. Saposin A (blue) is mixed together with HEGA-10 and VLPs containing HIV-1 proteins such as, gp120 subunit (brown), gp41 subunit (grey), CA protein (white circle) and MA protein (green). HEGA-10 is then removed using two subsequent spin SEC columns (Molecular weight cut-off 7 kDa) and the Salipro-HIV-spike particles are purified using a lectin affinity column. The lectin binds to high mannose sugar moieties on the spike protein. The column was washed with 50 column volumes HN buffer followed by elution using 0.3 M methyl-αD-mannopyranoside.

Supplementary Figure 6 Optimization of the amount of saposin A for efficient Salipro–HIV spike nanoparticle formation.

Radioactively labeled VLPs were mixed with Saposin A (230-0.77 μg/ml) followed by 10 min solubilisation on ice using 9 mM HEGA-10 in 1x HNC buffer. HEGA-10 was then removed using a SEC spin column and the amount of reconstituted Salipro-HIV-spike particles was monitored by BN-PAGE. About 100 μg/ml Saposin A was found to be optimal.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Table 1 (PDF 694 kb)

Cryo-EM structure of Salipro-POT.

3D density map of the tetrameric bacterial transporter with each of the subunits and tentative placement of Saposin (orange and blue, respectively) in the density. (MOV 23680 kb)

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Frauenfeld, J., Löving, R., Armache, JP. et al. A saposin-lipoprotein nanoparticle system for membrane proteins. Nat Methods 13, 345–351 (2016). https://doi.org/10.1038/nmeth.3801

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