Flexible, symmetry-directed approach to assembling protein cages

Abstract

The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.

Keywords: analytical ultracentrifugation; coiled coils; cryoelectron microscopy; native mass spectrometry; protein design.

Fig. 1.

Design of a self-assembling octahedral protein cage. (A) Structures of the trimeric esterase (PDB 1ZOI) (C termini of the esterase are indicated by red spheres) and the tetrameric coiled coil (PDB 3R4A) used in the design. (B) Minimization of linker distance compatible with octahedral geometry. The proteins were arrayed along the C₃ (blue line) and C₄ (green line) symmetry axes, and the distance between the N terminus of the coiled coil and the C terminus of the esterase (dashed red line) was minimized by symmetrically varying the rotation of the proteins about the symmetry axes and their radial distance while avoiding steric clashes. (C) Distance-minimized structures were found to be compatible with the coiled-coil domains either facing inward (top structure) or outward (bottom structure) with a minimum interterminus distance of ∼9.1 Å.

Fig. 2.

Initial characterization of Oct-2 and Oct-4. (A) SEC of Oct-4, Oct-2, and the unmodified esterase. (B) Native gel electrophoresis of Oct-4, Oct-2, and the unmodified esterase.

Fig. S1.

(A) SDS PAGE of proteins. Lane 1, protein standards; lane 2, unmodified esterase; lane 3, Oct-4; and lane 4, Oct-2. (B, Left) SEC of Oct-4 after purification on Ni-NTA resin (solid trace). Fractions 1–5 were analyzed by native PAGE, pooled, and rechromatographed (dashed trace). (Right) Analysis of SEC fractions by native PAGE. Lanes on the gel are: Ni, Oct-4 after purification on Ni-NTA resin; lanes 1–5, fractions 1–5; and pool, pooled material after SEC.

Fig. 3.

Structural characterization of Oct-4. (A) Sedimentation velocity AUC of Oct-4. The protein sediments primarily (>75%) as a single, well-defined species with an appropriate weight and shape for a 24-subunit octahedron. (B) Native electrospray mass spectrum of intact Oct-4. The envelope of charge states centered at m/z 12,600 corresponds to a species of M_r = 887 ± 5 kDa, whereas those centered at m/z = 11200 corresponds to a species of M_r = 757 ± 7 kDa. The smaller species represents dissociation of one trimer from the octahedral complex under the conditions of the Native MS experiment. (C) Negative stain EM images of the particles formed by Oct-4. Arrows indicate particles where fourfold symmetry is apparent. (Scale bar, 20 nm.) (Inset) Negative stain EM of unmodified trimeric esterase. (D, Left) Representative 2D class-averaged images of Oct-4 and projections generated from the 3D electron density map. (Right) Reconstructed electron density for Oct-4 viewed along the fourfold and threefold axes with one esterase trimer shown modeled into the electron density. The lower images show a slice through the electron density.

Fig. S2.

Further characterization of Oct-2. (A) A 2DSA of Oct-2. The protein forms multiple species characterized by sedimentation coefficients that are larger than expected for an octahedral cage. The low frictional ratios are consistent with the formation of globular complexes. (B) Negative stain EM of Oct-2. The images indicate that the protein assembles into a range of particle sizes, but no symmetry is apparent in the images, in contrast to the particles formed by Oct-4 (Fig. 3D). (Scale bar, 20 nm.)

Fig. S3.

The 2D class averages for Oct-4 from cryo-EM. A total of 44,856 particle images representing protein cages were excised using RELION. The selected particles were further subjected to reference-free alignment and classified into 405 classes. For details, see The 2D Classifications.

Fig. S4.

(A) Initial electron density model used in 3D reconstruction of Oct-4 from cryo-EM data. Model is shown viewed along threefold and fourfold symmetry axes. (B) Estimation of resolution of the reconstructed model of Oct-4. The final map of the protein cage was produced with an indicated resolution of 17 Å at the 0.5 level of Fourier shell correlation.