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.

Coiled coils unspring protein origami

“…Supercharging, introduced in the present report 1 , is the key to overcoming folding bottlenecks. The result is a computational platform for coiled-coil origami design that takes the desired geometry as input and outputs several complete sequences composed of unique coiled-coil-forming segments, along with a three-dimensional model of the design.…”

“…As a proponent of ‘functional design’, the minimalist architect Ludwig Mies van der Rohe believed that “Architecture starts when you carefully put two bricks together.” In this issue, Ljubetič et al 1 use α-helices as bricks to achieve architectural integrity with simplicity in protein design. By placing α-helices along an amino acid chain in such a way that they form coiled coils with cognate α-helices far apart in the sequence, the authors create hollow triangular pyramids, square pyramids, and triangular prisms that match predetermined shapes.…”

“…Ljubetič et al 1 were inspired by the similarities between DNA and coiled coils and by recent advances in DNA origami, in which strands of DNA are folded via Watson–Crick base pairing into a large variety of 2D and 3D structures 7 . The essence of origami is to make complex shapes by folding a single continuous entity.…”

“…DNA origami is usually composed of DNA alone, without added functional moieties, and the coiled-coil protein origami demonstrated by Ljubetič et al 1 is similarly minimalist. But as amino acids have chemically diverse side chains, many opportunities exist to add functionality and put muscle on the bones.…”

Artificial molecular and nanostructures for advanced nanomachinery

“…[84][85][86] Natural peptide chains self-assemble to form enzymes, some of which have been recently shown to exhibit nanomotor behaviours. 87 Enzymes are formed via the self-assembly of peptide chains.…”