Design of a hyperstable 60-subunit protein icosahedron

Journal name:
Nature
Volume:
535,
Pages:
136–139
Date published:
DOI:
doi:10.1038/nature18010
Received
Accepted
Published online
Corrected online

The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport1, 2. There has been considerable interest in repurposing such structures3, 4, 5 for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery6, vaccine design7 and synthetic biology8.

At a glance

Figures

  1. Design methodology and biochemical characterization.
    Figure 1: Design methodology and biochemical characterization.

    a, b, Icosahedral three-fold axis in red and aligned trimeric building block in green. c, Optimization of r and ω yields closely opposed interfaces between subunits. d, Sequence design yields low-energy interfaces; in the I3-01 case, composed of five designed residues (thick representations) and two native residues (thin representations). e, I3-01 appears larger by SEC than the similarly sized I3-01(L33R) and wild-type trimer (1wa3). f, DLS measurement of hydrodynamic radius (note logarithmic scale in f and h) of 1wa3 (3.5 nm) and I3-01 (14 nm). I3-01 remains assembled in 6.7 M GuHCl and in 2 M GITC. g, Extremely sharp disassociation to trimeric building blocks at 2.25 M GITC. Data points represent independent measurements. h, I3-01 icosahedron disassembles into the trimeric building blocks at 3 M GITC, and reassembles following dilution to 1 M.

  2. Cryo-EM.
    Figure 2: Cryo-EM.

    a, Field-of-view cryo-EM micrograph showing homogeneous icosahedral particles in various orientations. b, Back-projections of I3-01 from the design model. c, Cryo-EM class averages closely match the design projections along all three symmetry axes. d, e, The calculated initial, unrefined density (blue, 3.22σ) closely matches the design model (green).

  3. Tuning nanocage structure and function with genetic fusions.
    Figure 3: Tuning nanocage structure and function with genetic fusions.

    a, The left panel shows a cryo-EM micrograph of I3-01(ctGFP); the top right panel shows a computational model with sfGFP in green; the bottom right panel shows the class average along the five-fold axis. b, Fluorescence microscopy fields of view. c, Fluorescence intensity histograms. AFU, arbitrary fluorescence units; ± standard deviation. d, Correlation between the mean fluorescence intensity and sfGFP copy number for nanoparticles with different numbers of fused sfGFP molecules. Error bars are s.e.m. (n = 3). e, f, Computational model and class averages along the five-fold axis of negatively stained I3-01 (e) and I3-01(HB) (f); the helical bundle is shown in red. Weak density in the centre of the pentameric faces in I3-01 may reflect randomly packaged material. There is clear density in the centre of the pentameric faces in the I3-01(HB) class averages consistent with the model.

  4. I3-01 tolerance to temperature.
    Extended Data Fig. 1: I3-01 tolerance to temperature.

    DLS measurements as I3-01 is subjected to heating to 90 °C (solid line), then cooling to 25 °C (dotted line) in TBS (a), 6.7 M GuHCl (b) and 2 M GITC (c). Under all three conditions, any indications of aggregation or increase in size due to temperature appear to be completely reversible.

  5. Reproducibility of I3-01 transition in 2 M to 2.25 M GITC.
    Extended Data Fig. 2: Reproducibility of I3-01 transition in 2 M to 2.25 M GITC.

    Four examples each of independent measurements at 2 M (blue) and 2.25 M (red) GITC using DLS show the reproducibility of the cage disassociation. Histograms are plotted offset by 1% intensity from each other for clarity.

  6. SEC of T33-21 and I3-01 fused with sfGFP.
    Extended Data Fig. 3: SEC of T33-21 and I3-01 fused with sfGFP.

    Size exclusion chromatography traces for T33-21 (12mer in red and 24mer in blue) and I3-01 (60mer in green and 120mer in purple) sfGFP fusions, display increased particle sizes with increasing copies of GFP, but retain monodispersed populations. The N-terminal fusion of sfGFP (dashed line) is expected to extend mostly outward from the icosahedron, thus greatly increasing the hydrodynamic radius while the C-terminal fusion is predicted to occupy the internal void space. A230, ultraviolet absorbance at 230 nm; mAU, milli-absorbance units.

  7. Tolerance of I3-01–sfGFP fusions to GuHCl.
    Extended Data Fig. 4: Tolerance of I3-01–sfGFP fusions to GuHCl.

    N-terminal (red) and C-terminal (blue) sfGFP fusions were equilibrated to 0–6.4 M GuHCl. Ultraviolet absorbance at 490 nm (A490) monitors the unfolding of sfGFP (top, solid line and crosses). DLS experiments (top, dotted line and dots) reveal as sfGFP unfolds, the hydrodynamic radius increases slightly, and then stabilizes. The bottom panels show that in 1 M GuHCl (solid line) and in 6 M GuHCl (dotted line), the icosahedral assemblies remain relatively monodisperse.

  8. I3-01 C-terminal fusions with other fluorescent proteins.
    Extended Data Fig. 5: I3-01 C-terminal fusions with other fluorescent proteins.

    Fluorescent proteins mTurquoise2 (in blue) or sYFP2 (in green) were fused to the C terminus of I3-01. The field of view using widefield fluorescence microscopy shows distinct signals of each type when the two types are mixed together.

  9. I3-01 retains native enzyme activity.
    Extended Data Fig. 6: I3-01 retains native enzyme activity.

    Coupled KDPG aldolase assay showing native-like enzymatic activity in I3-01. The K129A knockout shows no enzyme activity, similar to buffer alone. UV339, absorbance at 339 nm; error bars are standard deviation.

Change history

Corrected online 06 July 2016
An addition was made to the Acknowledgements section.

References

  1. Zandi, R., Reguera, D., Bruinsma, R. F., Gelbart, W. M. & Rudnick, J. Origin of icosahedral symmetry in viruses. Proc. Natl Acad. Sci. USA 101, 1555615560 (2004)
  2. Ritsert, K. et al. Studies on the lumazine synthase/riboflavin synthase complex of Bacillus subtilis: crystal structure analysis of reconstituted, icosahedral beta-subunit capsids with bound substrate analogue inhibitor at 2.4 Å resolution. J. Mol. Biol. 253, 151167 (1995)
  3. Howorka, S. Rationally engineering natural protein assemblies in nanobiotechnology. Curr. Opin. Biotechnol. 22, 485491 (2011)
  4. Roldão, A., Mellado, M. C. M., Castilho, L. R., Carrondo, M. J. T. & Alves, P. M. Virus-like particles in vaccine development. Expert Rev. Vaccines 9, 11491176 (2010)
  5. Effio, C. L. & Hubbuch, J. Next generation vaccines and vectors: designing downstream processes for recombinant protein-based virus-like particles. Biotechnol. J. 10, 715727 (2015)
  6. Ma, Y., Nolte, R. J. M. & Cornelissen, J. J. L. M. Virus-based nanocarriers for drug delivery. Adv. Drug Deliv. Rev. 64, 811825 (2012)
  7. Smith, M. L. et al. Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications. Virology 348, 475488 (2006)
  8. Bauler, P., Huber, G., Leyh, T. & McCammon, J. A. Channeling by proximity: the catalytic advantages of active site colocalization using Brownian dynamics. J. Phys. Chem. Lett. 1, 13321335 (2010)
  9. Brodin, J. D. et al. Metal-directed, chemically tunable assembly of one-, two- and three-dimensional crystalline protein arrays. Nat. Chem. 4, 375382 (2012)
  10. Der, B. S. et al. Metal-mediated affinity and orientation specificity in a computationally designed protein homodimer. J. Am. Chem. Soc. 134, 375385 (2012)
  11. Fletcher, J. M. et al. Self-assembling cages from coiled-coil peptide modules. Science 340, 595599 (2013)
  12. Usui, K. et al. Nanoscale elongating control of the self-assembled protein filament with the cysteine-introduced building blocks. Protein Sci. 18, 960969 (2009)
  13. Raman, S., Machaidze, G., Lustig, A., Aebi, U. & Burkhard, P. Structure-based design of peptides that self-assemble into regular polyhedral nanoparticles. Nanomedicine 2, 95102 (2006)
  14. Raman, S. et al. Design of peptide nanoparticles using simple protein oligomerization domains. Open Nanomed. J. 2, 1526 (2009)
  15. Sinclair, J. C., Davies, K. M., Vénien-Bryan, C. & Noble, M. E. M. Generation of protein lattices by fusing proteins with matching rotational symmetry. Nat. Nanotechnol. 6, 558562 (2011)
  16. Boyle, A. L. et al. Squaring the circle in peptide assembly: from fibers to discrete nanostructures by de novo design. J. Am. Chem. Soc. 134, 1545715467 (2012)
  17. Lai, Y.-T. et al. Structure of a designed protein cage that self-assembles into a highly porous cube. Nat. Chem. 6, 10651071 (2014)
  18. King, N. P. et al. Computational design of self-assembling protein nanomaterials with atomic level accuracy. Science 336, 11711174 (2012)
  19. King, N. P. et al. Accurate design of co-assembling multi-component protein nanomaterials. Nature 510, 103108 (2014)
  20. Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 487, 545574 (2011)
  21. DiMaio, F., Leaver-Fay, A., Bradley, P., Baker, D. & André, I. Modeling symmetric macromolecular structures in Rosetta3. PLoS One 6, e20450 (2011)
  22. Lawrence, M. C. & Colman, P. M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946950 (1993)
  23. Griffiths, J. S. et al. Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase. Bioorg. Med. Chem. 10, 545550 (2002)
  24. Fullerton, S. W. B. et al. Mechanism of the class I KDPG aldolase. Bioorg. Med. Chem. 14, 30023010 (2006)
  25. Perlmutter, J. D. & Hagan, M. F. Mechanisms of virus assembly. Annu. Rev. Phys. Chem. 66, 217239 (2015)
  26. Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 7988 (2006)
  27. Andrews, B. T., Schoenfish, A. R., Roy, M., Waldo, G. & Jennings, P. A. The rough energy landscape of superfolder GFP is linked to the chromophore. J. Mol. Biol. 373, 476490 (2007)
  28. Cortese, K., Diaspro, A. & Tacchetti, C. Advanced correlative light/electron microscopy: current methods and new developments using Tokuyasu cryosections. J. Histochem. Cytochem. 57, 11031112 (2009)
  29. Huang, P.-S. et al. High thermodynamic stability of parametrically designed helical bundles. Science 346, 481485 (2014)
  30. Zhou, Z. et al. Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases. ACS Chem. Biol. 2, 337346 (2007)
  31. Baalousha, M. & Lead, J. R. Nanoparticle dispersity in toxicology. Nat. Nanotechnol. 8, 308309 (2013)
  32. Zulauf, M. & D’Arcy, A. Light scattering of proteins as a criterion for crystallization. J. Cryst. Growth 122, 102106 (1992)
  33. Nannenga, B. L., Iadanza, M. G., Vollmar, B. S. & Gonen, T. in Current Protocols in Protein Science (eds Coligan, J. E., Dunn, B. M., Speicher, D. W. & Wingfield, P. T.) Ch. 17.15 (John Wiley & Sons, 2013)
  34. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 3846 (2007)
  35. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671675 (2012)
  36. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584590 (2013)
  37. van Heel, M., Harauz, G., Orlova, E. V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 1724 (1996)
  38. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 16051612 (2004)
  39. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190199 (1996)
  40. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676682 (2012)
  41. Huang, P.-S. et al. RosettaRemodel: a generalized framework for flexible backbone protein design. PLoS One 6, e24109 (2011)
  42. Muller, E. G. D. et al. The organization of the core proteins of the yeast spindle pole body. Mol. Biol. Cell 16, 33413352 (2005)
  43. Shimogawa, M. M., Wargacki, M. M., Muller, E. G. & Davis, T. N. Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint. Cell Cycle 9, 36193628 (2010)

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Author information

  1. These authors contributed equally to this work.

    • Yang Hsia &
    • Jacob B. Bale

Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA

    • Yang Hsia,
    • Jacob B. Bale,
    • Shane Gonen,
    • William Sheffler,
    • Kimberly K. Fong,
    • Una Nattermann,
    • Chunfu Xu,
    • Po-Ssu Huang,
    • Rashmi Ravichandran,
    • Sue Yi,
    • Trisha N. Davis,
    • Neil P. King &
    • David Baker
  2. Institute for Protein Design, University of Washington, Seattle, Washington 98195, USA

    • Yang Hsia,
    • Jacob B. Bale,
    • Shane Gonen,
    • William Sheffler,
    • Una Nattermann,
    • Chunfu Xu,
    • Po-Ssu Huang,
    • Rashmi Ravichandran,
    • Sue Yi,
    • Neil P. King &
    • David Baker
  3. Graduate Program in Biological Physics, Structure and Design, University of Washington, Seattle, Washington 98195, USA

    • Yang Hsia,
    • Shane Gonen &
    • Una Nattermann
  4. Graduate Program in Molecular and Cellular Biology, University of Washington, Seattle, Washington 98195, USA

    • Jacob B. Bale
  5. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA

    • Shane Gonen,
    • Dan Shi &
    • Tamir Gonen
  6. Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA

    • David Baker

Contributions

J.B.B., N.P.K., and W.S. developed the computational design methodology. Y.H. and J.B.B. performed the design of the icosahedra. Y.H. performed all other unlisted experiments. S.G. and D.S. performed the cryo-EM experiments. K.K.F. performed the fluorescence microscopy experiments. U.N. performed the negative-stain electron microscopy experiments. C.X. provided the pentamer sequence for I3-01(HB). P.-S.H. created the computational methodology to model fusions to I3-01. R.R. produced I3-01(HB) proteins. S.Y. produced T33-21 sfGFP fusion proteins.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: I3-01 tolerance to temperature. (59 KB)

    DLS measurements as I3-01 is subjected to heating to 90 °C (solid line), then cooling to 25 °C (dotted line) in TBS (a), 6.7 M GuHCl (b) and 2 M GITC (c). Under all three conditions, any indications of aggregation or increase in size due to temperature appear to be completely reversible.

  2. Extended Data Figure 2: Reproducibility of I3-01 transition in 2 M to 2.25 M GITC. (178 KB)

    Four examples each of independent measurements at 2 M (blue) and 2.25 M (red) GITC using DLS show the reproducibility of the cage disassociation. Histograms are plotted offset by 1% intensity from each other for clarity.

  3. Extended Data Figure 3: SEC of T33-21 and I3-01 fused with sfGFP. (175 KB)

    Size exclusion chromatography traces for T33-21 (12mer in red and 24mer in blue) and I3-01 (60mer in green and 120mer in purple) sfGFP fusions, display increased particle sizes with increasing copies of GFP, but retain monodispersed populations. The N-terminal fusion of sfGFP (dashed line) is expected to extend mostly outward from the icosahedron, thus greatly increasing the hydrodynamic radius while the C-terminal fusion is predicted to occupy the internal void space. A230, ultraviolet absorbance at 230 nm; mAU, milli-absorbance units.

  4. Extended Data Figure 4: Tolerance of I3-01–sfGFP fusions to GuHCl. (198 KB)

    N-terminal (red) and C-terminal (blue) sfGFP fusions were equilibrated to 0–6.4 M GuHCl. Ultraviolet absorbance at 490 nm (A490) monitors the unfolding of sfGFP (top, solid line and crosses). DLS experiments (top, dotted line and dots) reveal as sfGFP unfolds, the hydrodynamic radius increases slightly, and then stabilizes. The bottom panels show that in 1 M GuHCl (solid line) and in 6 M GuHCl (dotted line), the icosahedral assemblies remain relatively monodisperse.

  5. Extended Data Figure 5: I3-01 C-terminal fusions with other fluorescent proteins. (490 KB)

    Fluorescent proteins mTurquoise2 (in blue) or sYFP2 (in green) were fused to the C terminus of I3-01. The field of view using widefield fluorescence microscopy shows distinct signals of each type when the two types are mixed together.

  6. Extended Data Figure 6: I3-01 retains native enzyme activity. (108 KB)

    Coupled KDPG aldolase assay showing native-like enzymatic activity in I3-01. The K129A knockout shows no enzyme activity, similar to buffer alone. UV339, absorbance at 339 nm; error bars are standard deviation.

Supplementary information

Zip files

  1. Supplementary Information (3.4 MB)

    This zipped file contains the protein sequences, the design structure PDB file and an example script.

Comments

  1. Report this comment #68233

    Howard Wilk said:

    Those are not icosahedra; they're dodecahedra. A regular icosahedron has 20 faces and 12 vertices. The regular dodecahedron has 12 faces and 20 vertices. The two polyhedra are duals of each other, so the symmetry is the same.

    Polyhedra are named after the number of their faces. Those nanocages have 12 faces and 20 vertices. You see 20-something, trimers or otherwise, you think icosa – 20 – hedron. I have to catch myself from making the error all the time.

    For another chemical example, look up "dodecahedrane".

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