Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo

Abstract

Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized >20 single-chain protein cages in three shapes—tetrahedron, four-sided pyramid, and triangular prism—with the largest containing >700 amino-acid residues and measuring 11 nm in diameter. Their stability and folding kinetics were similar to those of natural proteins. Solution small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophysical analysis confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: CC module structure and CCPO nomenclature.
Figure 2: Design and biophysical analysis of two tetrahedral structures.
Figure 3: Coiled-coil protein-origami design platform (CoCoPOD).
Figure 4: Design and biophysical analysis of the four-sided pyramid and triangular prism protein-origami folds.
Figure 5: Structural characterization by solution SAXS.
Figure 6: Protein origami folds in mammalian cells and in mice.

Accession codes

Primary accessions

Electron Microscopy Data Bank

References

  1. 1

    Douglas, S.M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Seeman, N.C. Nanomaterials based on DNA. Annu. Rev. Biochem. 79, 65–87 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    He, Y. et al. Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra. Nature 452, 198–201 (2008).

    Article  CAS  Google Scholar 

  4. 4

    Veneziano, R. et al. Designer nanoscale DNA assemblies programmed from the top down. Science 352, 1534 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Benson, E. et al. DNA rendering of polyhedral meshes at the nanoscale. Nature 523, 441–444 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Lupas, A.N. & Alva, V. Ribosomal proteins as documents of the transition from unstructured (poly)peptides to folded proteins. J. Struct. Biol. 198, 74–81 (2017).

    Article  CAS  Google Scholar 

  7. 7

    Demain, A.L. & Vaishnav, P. Production of recombinant proteins by microbes and higher organisms. Biotechnol. Adv. 27, 297–306 (2009).

    Article  CAS  Google Scholar 

  8. 8

    Taylor, W.R., Chelliah, V., Hollup, S.M., MacDonald, J.T. & Jonassen, I. Probing the “dark matter” of protein fold space. Structure 17, 1244–1252 (2009).

    Article  CAS  Google Scholar 

  9. 9

    Kuhlman, B. et al. Design of a novel globular protein fold with atomic-level accuracy. Science 302, 1364–1368 (2003).

    Article  CAS  Google Scholar 

  10. 10

    King, N.P. et al. Computational design of self-assembling protein nanomaterials with atomic level accuracy. Science 336, 1171–1174 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Doyle, L. et al. Rational design of α-helical tandem repeat proteins with closed architectures. Nature 528, 585–588 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Regan, L. & DeGrado, W.F. Characterization of a helical protein designed from first principles. Science 241, 976–978 (1988).

    Article  CAS  Google Scholar 

  13. 13

    Woolfson, D.N. The design of coiled-coil structures and assemblies. Adv. Protein Chem. 70, 79–112 (2005).

    Article  CAS  Google Scholar 

  14. 14

    Gradišar, H. et al. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments. Nat. Chem. Biol. 9, 362–366 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Plückthun, A. Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy. Annu. Rev. Pharmacol. Toxicol. 55, 489–511 (2015).

    Article  CAS  Google Scholar 

  16. 16

    Grove, T.Z., Cortajarena, A.L. & Regan, L. Ligand binding by repeat proteins: natural and designed. Curr. Opin. Struct. Biol. 18, 507–515 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Bella, J., Hindle, K.L., McEwan, P.A. & Lovell, S.C. The leucine-rich repeat structure. Cell. Mol. Life Sci. 65, 2307–2333 (2008).

    Article  CAS  Google Scholar 

  18. 18

    Pinheiro, A.V., Han, D., Shih, W.M. & Yan, H. Challenges and opportunities for structural DNA nanotechnology. Nat. Nanotechnol. 6, 763–772 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Kočar, V. et al. Design principles for rapid folding of knotted DNA nanostructures. Nat. Commun. 7, 10803 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Drobnak, I., Gradišar, H., Ljubetič, A., Merljak, E. & Jerala, R. Modulation of Coiled-Coil Dimer Stability through Surface Residues while Preserving Pairing Specificity. J. Am. Chem. Soc. 139, 8229–8236 (2017).

    Article  CAS  Google Scholar 

  21. 21

    Götze, M. et al. StavroX--a software for analyzing cross-linked products in protein interaction studies. J. Am. Soc. Mass Spectrom. 23, 76–87 (2012).

    Article  CAS  Google Scholar 

  22. 22

    Lawrence, M.S., Phillips, K.J. & Liu, D.R. Supercharging proteins can impart unusual resilience. J. Am. Chem. Soc. 129, 10110–10112 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Plaxco, K.W., Simons, K.T. & Baker, D. Contact order, transition state placement and the refolding rates of single domain proteins. J. Mol. Biol. 277, 985–994 (1998).

    Article  CAS  Google Scholar 

  24. 24

    Kočar, V. et al. TOPOFOLD, the designed modular biomolecular folds: polypeptide-based molecular origami nanostructures following the footsteps of DNA. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 7, 218–237 (2015).

    Article  CAS  Google Scholar 

  25. 25

    Negron, C. & Keating, A.E. A set of computationally designed orthogonal antiparallel homodimers that expands the synthetic coiled-coil toolkit. J. Am. Chem. Soc. 136, 16544–16556 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Doig, A.J. & Baldwin, R.L. N- and C-capping preferences for all 20 amino acids in alpha-helical peptides. Protein Sci. 4, 1325–1336 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Fijavž, G., Pisanski, T. & Rus, J. Strong traces model of self-assembly polypeptide structures. MATCH Commun. Math. Comput. Chem. 71, 199–212 (2014)<.

    Google Scholar 

  28. 28

    Noel, J.K. et al. SMOG 2: a versatile software package for generating structure-based models. PLoS Comput. Biol. 12, e1004794 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Englander, S.W. & Mayne, L. The nature of protein folding pathways. Proc. Natl. Acad. Sci. USA 111, 15873–15880 (2014).

    Article  CAS  Google Scholar 

  30. 30

    Agyemang, A.F., Harrison, S.R., Siegel, R.M. & McDermott, M.F. Protein misfolding and dysregulated protein homeostasis in autoinflammatory diseases and beyond. Semin. Immunopathol. 37, 335–347 (2015).

    Article  CAS  Google Scholar 

  31. 31

    Jahn, K. et al. Functional patterning of DNA origami by parallel enzymatic modification. Bioconjug. Chem. 22, 819–823 (2011).

    Article  CAS  Google Scholar 

  32. 32

    Padilla, J.E., Colovos, C. & Yeates, T.O. Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Proc. Natl. Acad. Sci. USA 98, 2217–2221 (2001).

    Article  CAS  Google Scholar 

  33. 33

    Lai, Y.-T. et al. Designing and defining dynamic protein cage nanoassemblies in solution. Sci. Adv. 2, e1501855 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    King, N.P. et al. Accurate design of co-assembling multi-component protein nanomaterials. Nature 510, 103–108 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Hsia, Y. et al. Design of a hyperstable 60-subunit protein icosahedron. Nature 535, 136–139 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Fletcher, J.M. et al. Self-assembling cages from coiled-coil peptide modules. Science 340, 595–599 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Wen, A.M. & Steinmetz, N.F. Design of virus-based nanomaterials for medicine, biotechnology, and energy. Chem. Soc. Rev. 45, 4074–4126 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Brunette, T.J. et al. Exploring the repeat protein universe through computational protein design. Nature 528, 580–584 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Kanekiyo, M. et al. Rational design of an Epstein-Barr virus vaccine targeting the receptor-binding site. Cell 162, 1090–1100 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    López-Sagaseta, J., Malito, E., Rappuoli, R. & Bottomley, M.J. Self-assembling protein nanoparticles in the design of vaccines. Comput. Struct. Biotechnol. J. 14, 58–68 (2016).

    Article  CAS  Google Scholar 

  41. 41

    Kushnir, N., Streatfield, S.J. & Yusibov, V. Virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development. Vaccine 31, 58–83 (2012).

    Article  CAS  Google Scholar 

  42. 42

    Correia, B.E. et al. Proof of principle for epitope-focused vaccine design. Nature 507, 201–206 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Kanekiyo, M. et al. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 499, 102–106 (2013).

    Article  CAS  Google Scholar 

  44. 44

    Eswar, N. et al. Comparative protein structure modeling using MODELLER. Curr. Protoc. Protein Sci. 50, 2.9.1–2.9.31 (2007).

    Article  Google Scholar 

  45. 45

    Pettersen, E.F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  PubMed  Google Scholar 

  46. 46

    McGibbon, R.T. et al. MDTraj: a modern open library for the analysis of molecular dynamics trajectories. Biophys. J. 109, 1528–1532 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Perez, F. & Granger, B.E. IPython: a system for interactive scientific computing. Comput. Sci. Eng. 9, 21–29 (2007).

    Article  CAS  Google Scholar 

  48. 48

    Ivankov, D.N. et al. Contact order revisited: influence of protein size on the folding rate. Protein Sci. 12, 2057–2062 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Testa, O.D., Moutevelis, E. & Woolfson, D.N. CC+: a relational database of coiled-coil structures. Nucleic Acids Res. 37, D315–D322 (2009).

    Article  CAS  Google Scholar 

  50. 50

    Grigoryan, G., Reinke, A.W. & Keating, A.E. Design of protein-interaction specificity gives selective bZIP-binding peptides. Nature 458, 859–864 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Gradišar, H. & Jerala, R. De novo design of orthogonal peptide pairs forming parallel coiled-coil heterodimers. J. Pept. Sci. 17, 100–106 (2011).

    Article  CAS  Google Scholar 

  52. 52

    Zhao, X., Ghaffari, S., Lodish, H., Malashkevich, V.N. & Kim, P.S. Structure of the Bcr-Abl oncoprotein oligomerization domain. Nat. Struct. Biol. 9, 117–120 (2002).

    CAS  PubMed  Google Scholar 

  53. 53

    Oshaben, K.M., Salari, R., McCaslin, D.R., Chong, L.T. & Horne, W.S. The native GCN4 leucine-zipper domain does not uniquely specify a dimeric oligomerization state. Biochemistry 51, 9581–9591 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Wood, C.W. et al. CCBuilder: an interactive web-based tool for building, designing and assessing coiled-coil protein assemblies. Bioinformatics 30, 3029–3035 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Phillips, J.C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Abraham, M.J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 2, 1–7 (2015).

    Google Scholar 

  57. 57

    Chen, Y.H., Yang, J.T. & Chau, K.H. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry 13, 3350–3359 (1974).

    Article  CAS  Google Scholar 

  58. 58

    Schrödinger, LLC. The PyMOL Molecular Graphics System, Version1.8. (2015).

  59. 59

    Drobnak, I., Vesnaver, G. & Lah, J. Model-based thermodynamic analysis of reversible unfolding processes. J. Phys. Chem. B 114, 8713–8722 (2010).

    Article  CAS  Google Scholar 

  60. 60

    Press, W.H., Teukolsky, S.A., Vetterling, W.T. & Flannery, B.P. Numerical Recipes in C++: The Art of Scientific Computing (Cambridge University Press, 2002).

  61. 61

    Gough, B., ed. GNU Scientific Library Reference Manual (Network Theory Ltd., 2009).

  62. 62

    Konarev, P., Volkov, V., Sokolova, A., Koch, M. & Svergun, D. PRIMUS - a Windows-PC based system for small-angle scattering data analysis. J. Appl. Cryst. 36, 1277–1282 (2003).

    Article  CAS  Google Scholar 

  63. 63

    Förster, S., Apostol, L. & Bras, W. Scatter: software for the analysis of nano-and mesoscale small-angle scattering. J. Appl. Cryst. 43, 639–646 (2010).

    Article  CAS  Google Scholar 

  64. 64

    Franke, D. & Svergun, D.I. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Cryst. 42, 342–346 (2009).

    Article  CAS  Google Scholar 

  65. 65

    Hura, G.L. et al. Comprehensive macromolecular conformations mapped by quantitative SAXS analyses. Nat. Methods 10, 453–454 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. 66

    Schneidman-Duhovny, D., Hammel, M. & Sali, A. FoXS: a web server for rapid computation and fitting of SAXS profiles. Nucleic Acids Res. 38, W540–W544 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. 67

    Bakan, A., Meireles, L.M. & Bahar, I. ProDy: protein dynamics inferred from theory and experiments. Bioinformatics 27, 1575–1577 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. 68

    Petoukhov, M.V. et al. New developments in the ATSAS program package for small-angle scattering data analysis. J. Appl. Crystallogr. 45, 342–350 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. 69

    Shevchenko, A., Tomas, H., Havliš, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2007).

    Article  CAS  Google Scholar 

  70. 70

    de la Rosa-Trevín, J.M. et al. Scipion: a software framework toward integration, reproducibility and validation in 3D electron microscopy. J. Struct. Biol. 195, 93–99 (2016).

    Article  Google Scholar 

  71. 71

    Abrishami, V. et al. A pattern matching approach to the automatic selection of particles from low-contrast electron micrographs. Bioinformatics 29, 2460–2468 (2013).

    Article  CAS  Google Scholar 

  72. 72

    Sorzano, C.O.S. et al. A clustering approach to multireference alignment of single-particle projections in electron microscopy. J. Struct. Biol. 171, 197–206 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. 73

    Goddard, T.D., Huang, C.C. & Ferrin, T.E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007).

    Article  CAS  Google Scholar 

  74. 74

    Wang, Y. et al. Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J. Biol. Chem. 275, 27013–27020 (2000).

    CAS  PubMed  Google Scholar 

  75. 75

    Hornung, V. et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9, 847–856 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. 76

    Hafner-Bratkovič, I., Benčina, M., Fitzgerald, K.A., Golenbock, D. & Jerala, R. NLRP3 inflammasome activation in macrophage cell lines by prion protein fibrils as the source of IL-1β and neuronal toxicity. Cell. Mol. Life Sci. 69, 4215–4228 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by the Slovenian Research Agency, program P4-0176, projects N4-0037 and J4-5528 (R.J.), L4-6812 (H.G.), and J3-7034 and BI-US/17-18-051 (M.B.); the ERANET SynBio project Bioorigami (ERASYNBIO1-006 to R.J.); COST actions CM1304 (R.J. and A.L.) and CM1306 (R.J. and J.A.); a grant from ICGEB (CRP/SLO14-03) to H.G.; ESRF, for making available its facility for performing SAXS measurements; MSC-ETN 642157 Tollerant H2020 (R.J. and F.L.). This work has been supported by iNEXT, PID1771 (R.J.), PID2706 (R.J.), PID1824 (H.G.), VID3987 (H.G.), funded by the Horizon 2020 Programme of the EU; NVIDIA Corporation for the donation of the Quadro GP100 GPU (J.M.C.); and FP7 project FCUB ERA (GA No. 256716 to T.Ć.V.) for the use of the proteomics facility. We thank K. Djinović Carugo for useful suggestions and for performing and analyzing the initial SAXS experiments. We thank the staff of the Centre for Laboratory Animals at Biotechnical faculty of the University of Ljubljana, where animal experiments were performed. We would like to thank K. Butina, R. Bremšak, I. Škraba, D. Oven, T. Lončar, S. Božič Abram, T. Doles, S. Grudinin, J. Mihailović, and E. Žagar for their technical support. We also thank E. Žerovnik for granting access to the stopped-flow circular dichroism instrument and C. Wood for building preliminary models of the CC pairs. Plasmid encoding firefly luciferase under the ATF6 control (p5XATF6-GL3) was a gift from R. Prywes (Columbia University, New York, NY, USA). Immortalized mouse bone-marrow-derived macrophages were a gift from K. Fitzgerald (University of Massachusetts Medical School, Worcester, MA, USA).

Author information

Affiliations

Authors

Contributions

A.L., F.L., H.G., I.D., J.A., Ž.S., and R.J. designed the CCPO variants. F.L., H.G., Ž.S. and N.K. cloned, purified, and experimentally characterized the proteins. A.L., I.D., J.A., and T.P. wrote the CoCoPOD platform. A.M. and T.Ć.V. performed the cross-linking experiments. J.A. and A.R. performed the SAXS experiments and SAXS data analysis. I.H.-B. and M.B. performed the experiments on the cells. M.B. and D.L. performed confocal microcopy imaging. D.L. performed the animal experiments. R.M. and J.M.C. performed the EM experiments and data processing. R.J. conceived the study, led the research, and wrote the initial manuscript. All authors discussed the results and reviewed and contributed to the manuscript.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Discussion, Supplementary Figures 1–22, Supplementary Tables 1–7, and Supplementary Note (PDF 18500 kb)

Life Sciences Reporting Summary (PDF 129 kb)

Supplementary Data

Topologies circular permutations TCO (XLSX 86 kb)

Supplementary Code

Supplementary Source Code (ZIP 1243 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ljubetič, A., Lapenta, F., Gradišar, H. et al. Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo. Nat Biotechnol 35, 1094–1101 (2017). https://doi.org/10.1038/nbt.3994

Download citation

Further reading