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Design of a hyperstable 60-subunit protein icosahedron

Nature volume 535, pages 136–139 (2016)Cite this article

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A Corrigendum to this article was published on 19 October 2016

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Abstract

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.

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Figure 1: Design methodology and biochemical characterization.
Figure 2: Cryo-EM.
Figure 3: Tuning nanocage structure and function with genetic fusions.

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Acknowledgements

This work was supported by the Howard Hughes Medical Institute (D.B. and T.G.), the JRC visitor programme (S.G.), the National Science Foundation CHE-1332907 (D.B.), a UW/Hutch CCSG Pilot Award NCI 5 P30 CA015704-41 (D.B. and N.P.K.), Takeda Pharmaceutical Company (N.P.K.), the Bill and Melinda Gates Foundation OPP1120319 (D.B. and N.P.K.), the National Institutes of Health (NIH) P41 GM103533 (T.N.D.), the Defense Advanced Research Projects Agency (D.B. and N.P.K., grant no. W911NF-14-1-0162) and the Air Force Office of Scientific Research (AFOSR) AFOSR FA950-12-10112 (D.B.). Y.H. was supported in part by a NIH Molecular Biology Training Grant (T32GM008268). U.N. was supported in part by a PHS National Research Service Award (T32GM007270) from NIGMS. J.B.B. was supported in part by an NSF Graduate Research Fellowship (DGE-0718124). We thank the Janelia Research Campus Cryo-EM Facility and J. de la Cruz for their assistance with the Titan Krios.

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Author notes

  1. Yang Hsia and Jacob B. Bale: These authors contributed equally to this work.

Authors and Affiliations

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

    Yang Hsia, Shane Gonen & Una Nattermann

  4. Graduate Program in Molecular and Cellular Biology, University of Washington, Seattle, 98195, Washington, USA

    Jacob B. Bale

  5. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, 20147, Virginia, USA

    Shane Gonen, Dan Shi & Tamir Gonen

  6. Howard Hughes Medical Institute, University of Washington, Seattle, 98195, Washington, USA

    David Baker

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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.

Corresponding author

Correspondence to David Baker.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 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.

Extended Data Figure 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.

Extended Data Figure 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.

Extended Data Figure 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.

Extended Data Figure 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.

Extended Data Figure 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.

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Supplementary Information

This zipped file contains the protein sequences, the design structure PDB file and an example script. (ZIP 3559 kb)

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Hsia, Y., Bale, J., Gonen, S. et al. Design of a hyperstable 60-subunit protein icosahedron. Nature 535, 136–139 (2016). https://doi.org/10.1038/nature18010

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