Computational design of self-assembling protein nanomaterials with atomic level accuracy

Abstract

We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

Fig. 1

General approach to designing self-assembling protein nanomaterials. (A) First, a target symmetric architecture is chosen. Octahedral point group symmetry is used in this example; the threefold rotational axes are marked here by triangles and shown as black lines throughout. The dashed cube is shown to orient the viewer. A symmetric oligomer which shares an element of symmetry with the target architecture, here a C3 symmetric trimer (green), is selected as a building block. (B) Multiple copies of the building block are symmetrically arranged in the target architecture by aligning their shared symmetry axes. The pre-existing organization of the oligomeric building block fixes several (in this case four) rigid body degrees of freedom (DOFs). The two remaining DOFs, radial displacement (r) and axial rotation (ω), are indicated. (C) Symmetrical docking is performed by systematically varying the two DOFs (moves are applied symmetrically to all subunits) and computing the suitability of each configuration for interface design (red: more suitable; blue: less suitable). Points corresponding to the docked configurations in panels (B), in which the building blocks are not in contact, and (D), a highly complementary interface, are indicated. (E) Closer view of the interface in (D). The interface lies on an octahedral two-fold symmetry axis shown as a grey line. In all steps before interface design, only backbone (shown in cartoon) and carbon beta (shown in sticks) atoms are considered. (F) Sequence design calculations are used to create low-energy protein-protein interfaces that drive self-assembly of the desired material. Designed hydrogen bonds across the interface are indicated by dashed lines.

Fig. 2

Experimental characterization of O3-33, T3-08, and T3-10. (A) Native PAGE of fluorescently labeled (from left) 3n79-wt, O3-33, 3ftt-wt, and T3-08 in lysates. Bands corresponding to the designed octahedral (O3-33) and tetrahedral (T3-08) assemblies are indicated with asterisks. SEC chromatgrams of nickel-purified (B) O3-33, (C) 3n79-wt, (D) O3-33(Ala167Arg), (E) T3-08, (F) T3-10, (G) 3ftt-wt, and (H) T3-08(Ala52Gln) collectively demonstrate that the assembly of the designed proteins is a result of the designed interfaces.

Fig. 3

Structural characterization of O3-33. (A) A representative negative stain electron micrograph of O3-33. Selected particles (boxed in white) that resemble views of the design model along its 4-fold, 2-fold, and 3-fold rotational axes, shown in (B), are enlarged at right. (B) The O3-33 design model, depicted in ribbon format. Each trimeric building block is shown in a different color. (C) The density map from a 20 Å resolution cryo-EM reconstruction of O3-33 clearly recapitulates the architecture of the design model. (D) The crystal structure of O3-33 (R32 crystal form). Images in (B) to (D) are shown to scale along the three types of symmetry axes present in point group O. (E) The designed interface in O3-33, highlighting the close agreement between the crystal structure (green and magenta) and the design model (white). Oxygen atoms are red; nitrogens, blue. Hydrogen bonds between the building blocks are shown as yellow dashes, and an octahedral 2-fold rotational axis that passes through the interface is shown as a gray line. Residues in which substitution disrupted self-assembly (see Fig. S4) are labeled.

Fig. 4

Structural characterization of T3-10. (A) A representative negative stain electron micrograph of T3-10. At bottom, averages of the particles resemble views of the design model along its 2-fold and 3-fold rotational axes, shown in (B). (B) Backbone representation T3-08/T3-10 design model, depicted as in Fig. 3B. (C) The T3-10 crystal structure. Images in (B) and (C) are shown to scale along the two types of symmetry axes present in point group T. (D) The designed interface in T3-10, revealing the close agreement of the crystal structure (green and magenta) to the design model (white). A network of polar interactions observed in the crystal structure at the designed interface is indicated by yellow dashes. The interface is viewed along an indicated tetrahedral 2-fold rotational axis. Alanine 52, which when mutated to glutamine in T3-08 disrupts assembly of the designed material, is labeled.