Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids1,2 or proteins3,4,5, but strategies for engineering hybrid biological materials are only beginning to emerge6,7. Here we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as ‘enveloped protein nanocages’ (EPNs). Robust EPN biogenesis requires protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the endosomal sorting complexes required for transport (ESCRT) machinery8. A variety of synthetic proteins with these functional elements induce EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical analyses and cryo-electron microscopy reveal that one design, EPN-01, comprises small (~100 nm) vesicles containing multiple protein nanocages that closely match the structure of the designed 60-subunit self-assembling scaffold9. EPNs that incorporate the vesicular stomatitis viral glycoprotein can fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These results show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells.
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This work was supported in part by Deutsche Forschungsgemeinschaft (DFG) Fellowship VO 1836/1-1 (to J.V.), an NIH Molecular Biology Training Grant (T32GM008268) (Y.H.), a PHS National Research Service Award (T32GM007270) from NIGMS (U.N.), grants from the Bill & Melinda Gates Foundation (OPP1118840) and Defense Advanced Research Projects Agency (W911NF-14-1-0162 and W911NF-15-1-0645) (N.P.K.), and NIH grants RO1 AI 51174 and P50 082545 (W.I.S.). We thank P. Shen and J. McCullough for assistance and advice on cryo-EM experiments, M. Kay and D. Eckert and J. Marvin (University of Utah FACS Core) for help with BlaM assays, S. Carter and S. Magesrawan (Caltech) for assistance with Amira Software, L. Nikolova (University of Utah EM Core) for assistance with immunogold labelling experiments, M. Redd (University of Utah Cell Imaging Core) for assistance with confocal microscopy, A. Wargacki for cloning assistance, S. Hauschka for tissue culture assistance, and M. Lajoie, L. Stewart and D. Baker for helpful discussions.
Extended data figures
Shown is the raw charge density in different slices of the tomogram (first pass) and isosurface renderings (second pass) of individual protein nanocages (gold) and the limiting membrane of the EPN (green).
Shown are the raw charge density (grey, contoured at 4.5 σ) and a rigid body fitting of the I3-01 design model (dark green) plus one Nterminal amino acid (yellow) added to I3-01.