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


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.

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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.

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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).

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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.

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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).

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