Computational design of a self-assembling symmetrical β-propeller protein

Abstract

The modular structure of many protein families, such as β-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate forms. Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical β-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2-10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry.

Keywords: computational protein design; protein crystallography; protein evolution; self-assembly; β-propeller.

Fig. 1.

Computational design of a fully symmetric β-propeller. From the nonsymmetrical six-bladed 1RWL template protein (A), the sequences of each blade were aligned (B) and used for ancestral sequence reconstruction. For comparison, the final Pizza sequence is also shown on the bottom line of B. Blade 3 was identified as closest to the most probable ancestral sequence and was used for the generation of a sixfold symmetrical template protein using RosettaDock with C6 symmetry. From the scatter plot (C) of the docking scores versus the rmsds between the different solutions and the best scoring solution (D), it is clear that the higher the deviation from the six-bladed propeller fold the worse the docking score becomes. The ancestral sequences and three WT sequences were mapped onto the fully symmetrical template and scored using Rosetta (E). The green bars indicate the 1RWL sequence scores (blades 3, 4, and 5). The red bar indicates the top-scoring sequence (Pizza2-SR). The orange bar corresponds to the selected Pizza sequence, which is also depicted as a Cα trace in F, colored blue to red from the N to C terminus. The differences between the Pizza and the Pizza2-SR sequence are annotated in red in B and F.

Fig. 2.

Purification and characterization of the Pizza proteins. Each protein was purified using size-exclusion chromatography. The SEC chromatograms show that all of the Pizza proteins can be purified to homogeneity although, for Pizza8, Pizza9, and Pizza10, a second SEC run was required (indicated with an asterisk) (A). Pizza7 forms two monodisperse species, Pizza7.1 and Pizza7.2. SDS/PAGE confirms the identity and purity of the proteins (B). The calibrated SEC curve (black) fitted to four experimental points (shown as crosses) agrees well with the predicted size of each Pizza protein, assuming that these proteins form solution complexes assembled to give sixfold symmetric units (C). AUC sedimentation curves confirm that the molecular weights in solution of the complexes agree with the LCM prediction, with Pizza7 forming the largest complex (D). The same color coding for the Pizza proteins is used in B, C, and D.

Fig. 3.

Crystallographic structures of the Pizza proteins. X-ray crystallographic analysis of five Pizza proteins confirmed the expected quaternary structure in each case, showing a six-bladed propeller. One blade of Pizza7H is not visible in the electron-density maps. Superposition of the expected and experimental structures (bottom row) demonstrates close agreement with the backbone-rmsd as shown. The mutated residues in Pizza2-SR are depicted as spheres.

Fig. 4.

Differential scanning fluorimetry protein melting and CD spectroscopy curves of Pizza6 and the different Pizza7 species. The monomeric heat-treated Pizza7H has a sharp melting curve, essentially identical to that of Pizza6 (A). The Pizza7.1 LCM complex shows a biphasic curve that also peaks at 77 °C, corresponding to the melting of the single-chained six-bladed propeller unit. The monomeric Pizza7.2 is a different protein species that melts around 57 °C under the conditions used. RFU, relative fluorescence units. CD spectroscopy (B), however, indicates that all four proteins are folded (40% β-sheet, in agreement with the crystal structures).