Computational design of bifaceted protein nanomaterials

Abstract

Advances in computational methods have led to considerable progress in the design of protein nanomaterials. However, nearly all nanoparticles designed so far exhibit strict point group symmetry, which limits structural diversity and precludes anisotropic functionalization. Here we describe a computational strategy for designing multicomponent bifaceted protein nanomaterials with two distinctly addressable sides. The method centres on docking pseudosymmetric hetero-oligomeric building blocks in architectures with dihedral symmetry and designing an asymmetric protein-protein interface between them. We obtain an initial 30-subunit assembly with pseudo-D₅ symmetry and generate variants in which we alter the size and morphology of the bifaceted nanoparticles by designing extensions to one of the subunits. Functionalization of the two nanoparticle faces with protein minibinders enables the specific colocalization of two populations of polystyrene microparticles coated with the target protein receptors. The ability to accurately design anisotropic protein nanoparticles could be broadly useful in applications requiring the colocalization of distinct target moieties.

Fig. 1. Overview of bifaceted pD5 architecture and design approach.

a, A nanoparticle with icosahedral symmetry constructed from homotrimeric building blocks requires only a single (symmetric) designed interface and is isotropic overall. b, A nanoparticle with dihedral symmetry (D_n; in this example, n = 5) constructed from a single heterotrimeric building block requires two designed interfaces: an asymmetric interface around the n-fold symmetry axis and a symmetric interface along the dihedral two-fold axes. The assembly is anisotropic, but the two opposing faces are constructed from the same three subunits (labelled 1, 2 and 3) and are not independently addressable. c, An anisotropic D_n assembly can be converted to a bifaceted pseudo-D_n assembly by asymmetrizing the designed interface at the dihedral two-fold axes. The nanoparticle is constructed from two different heterotrimers that can be produced independently before nanoparticle assembly, rendering each of the six subunits uniquely addressable, even if subunits 2 + 6 and 3 + 5 are genetically identical. d, Schematic of the procedure for docking Crown_C5-1 substructures into (ABC)₁₀ assemblies with D₅ symmetry. The A, B and C subunits of Crown_C5-1 are labelled, and the rotational and translational degrees of freedom sampled during docking are indicated along the five-fold symmetry axis. e, An example bifaceted (ABC)₅–(ABD)₅ assembly after asymmetric C–D interface design. f, Details of an example asymmetric C–D interface. Positions featuring different amino acids in the C and D subunits are highlighted.

Fig. 2. In vitro assembly and structural characterization of pD5-14.

a, SEC traces of pD5-14 (ABC)₅, (ABD)₅ and (ABC)₅–(ABD)₅ assemblies. b, SDS-PAGE of SEC-purified assemblies from a. The locations of the A, B, C and D subunits, as well as those of marker bands, are indicated. c–e, nsEM of pD5-14 (ABC)₅ (c), (ABD)₅ (d) and (ABC)₅–(ABD)₅ (e) assemblies, including representative 2D class averages and rigid-body fits of the computational design models into 3D reconstructions. f, Cryo-EM 2D class averages of pD5-14 (ABC)₅–(ABD)₅. g, Left: 4.30-Å-resolution cryo-EM density map of pD5-14 (ABC)₅–(ABD)₅ viewed along two orthogonal axes. Right: overlay between the computational design model (grey) and the experimentally determined cryo-EM model (colours). h, Details of asymmetrically designed C–D interface. Scale bars, 50 nm (raw micrographs); 20 nm (2D class averages). Source data

Fig. 3. Fine tuning of bifaceted protein nanoparticle size and shape using RFdiffusion.

a, Schematic of the approach used to define target extended architectures. Left: the scissors indicate the loop in the C subunit that was ‘cut’. Right: target architectures were defined by translating and in one case rotating most of the (ABC)₅ substructure, leaving the designed asymmetric C–D interface intact. b, Schematic of the design process used to generate de novo C subunits for each extended architecture. c, Number of designs that passed AF2 filtering (pLDDT > 90; r.m.s.d. to design model < 1.5 Å) per number of added amino acid residues for each extended architecture.

Fig. 4. nsEM characterization of extended (ABC)₅–(ABD)₅ nanoparticles.

From top to bottom: representative assemblies extended by 25 Å, 50 Å, 75 Å and 100 Å, or extended by 50 Å and rotated by 25°. From left to right: computational design models, raw micrographs of (ABC)₅, raw micrographs of (ABC)₅ + (ABD)₅, 2D class averages of (ABC)₅ + (ABD)₅ showing representative top and side views, 3D reconstructions and design models fit into the 3D reconstructions. Side views of the design models and 3D reconstructions are shown for each particle, except for pD5_+50/25°-344, for which the side and top views are shown. Scale bars, 50 nm (raw micrographs); 20 nm (2D class averages). Source data

Fig. 5. Minibinder-functionalized bifaceted nanoparticles colocalize two distinct fluorescent polystyrene microparticle populations.

a, Schematic of the approach used to (1) reorient the N termini of the A, B and C subunits of pD5-14 to face towards the nanoparticle exterior and (2) independently functionalize each face of the resultant bifaceted nanoparticle with protein minibinders. The newly diffused α-helix is shown in dark grey, and the N and C termini are indicated by blue and red circles, respectively. b,c, SEC trace (b) and nsEM (c) of pD5-14_rd47 with Neo-2/15 and 41bb_mb1 genetically fused to the B components of the (ABC)₅ and (ABD)₅ components, respectively. Scale bars, 50 nm (raw micrograph); 20 nm (2D class averages). d, Binding of functionalized bifaceted nanoparticles to IL-2Rβ (left) and 4-1BB (right) ectodomains, measured by BLI. The legend indicates which minibinder is fused to each component in each bifaceted nanoparticle. e, Representative flow cytometry plots showing the colocalization of IL-2Rβ-coated Nile Red and 4-1BB-coated purple polystyrene particles by functionalized bifaceted nanoparticles. f, Quantitation of colocalization detected by flow cytometry. Each data point represents the mean of two technical replicates across three independent biological replicates (n = 3). Each bar depicts the mean value of the group, and the error bars depict the standard deviation. Statistical significance was calculated via paired one-way analysis of variance with Geisser–Greenhouse correction followed by Tukey’s multiple comparisons test, with individual variances computed for each comparison. Exact P values are reported in the plot. g, Representative fluorescence microscopy images showing the colocalization of IL-2Rβ-coated Nile Red and 4-1BB-coated purple polystyrene particles by functionalized bifaceted nanoparticles. The percentage of colocalized beads observed across 25 independent fields of view is displayed in the top-right corner of each image. Scale bar, 60 μm. Source data

Extended Data Fig. 1. AF2 structure prediction metrics of designed asymmetric C-D interface.

a, AF2 metrics for the asymmetric C-D interface designs. The structures of the five C-terminal helices of the C and D subunits, comprising the designed interface, were predicted and pLDDT and pAE were plotted against RMSD to the computational design models (post-ProteinMPNN). Metrics for designs from each of the five ProteinMPNN strategies used to design asymmetric interfaces are shown separately. Each point represents a prediction from one of five AF2 models used to evaluate each design. Designs that passed the first round of filtering and were evaluated for off-target homotypic interface formation are boxed in red. b, AF2 metrics for off-target C-C or D-D interface formation using sequences that passed the first round of filtering. Only data corresponding to the best (that is, lowest mean pAE interaction) off-target prediction for each design are shown. Gray lines connect data points for designs that were predicted to form the on-target C-D interface (a) and predicted to not form off-target C-C or D-D interfaces (b). The red line connects the on-target and off-target data points for pD5-14, which was experimentally verified to form exclusively (ABC)₅-(ABD)₅ complexes.

Extended Data Fig. 2. Size distribution and thermal stability of pD5-14 bifaceted nanoparticles.

a, DLS and measured hydrodynamic diameters of pD5-14 (ABC)₅, (ABD)₅, and (ABC)₅-(ABD)₅. b, Mass photometry of pD5-14 (ABC)₅ + (ABD)₅, showing an observed mass of 1151 kDa. The expected mass for the 30-subunit assembly is 1252 kDa. c, DLS measurements of pD5-14 (ABC)₅-(ABD)₅ at 25 °C and 95 °C. d, Nano differential scanning fluorimetry of (ABC)₅-(ABD)₅, plotted as the barycentric mean (BCM) of the emission spectrum during heating from 25–95 °C. The y axis spans the range of BCM values typically observed during protein denaturation, while the inset zooms in on the y axis to show the details of the data. e, Evaluation of thermal aggregation of (ABC)₅-(ABD)₅, measured by scattering intensity at 266 nm during heating from 25–95 °C. Thermal aggregation of an N1 influenza neuraminidase ectodomain (N1-CA09-WT)⁷⁴ is shown for comparison, while the inset zooms in on the y axis to show the details of the data (ABC)₅-(ABD)₅. Aggregation temperatures, as determined by UNcle Analysis Software, are indicated by dashed lines. Each panel contains a representative example of at least 3 independent measurements from at least 2 different sample purifications.

Extended Data Fig. 3. Details of cryo-EM data processing.

a, Denoised representative cryo-EM micrograph. b, Representative 2D class averages with scale bar showing particles from multiple view angles, exemplifying preference for ‘Side’ views over ‘Top’ views. c, Fourier Shell Correlation (FSC) plot of volume map, illustrating 4.30 Å resolution estimation at FSC 0.143 cutoff. d, View angle distribution plot, demonstrating spatial preference for ‘Side’ views over ‘Top’ views. e, Cryo-EM model docked into cryo-EM volume. f, Local resolution estimation (Å) of cryo-EM volume. Scale bars: 20 nm.

Extended Data Fig. 4. Representative RFdiffusion and AF2 outputs of de novo extensions.

a, Backbone structures of ABC heterotrimers output from RFdiffusion for different extension lengths. The images are oriented such that the two C-terminal helices of the C subunit that make up the pseudo-dihedral interface (pink) are at bottom. The de novo extensions that connect the C-terminal helices to the rest of the C subunit are colored gray. RFdiffusion generated diverse structures including both ɑ-helices and β-sheets. b, Representative extended ABC heterotrimers that passed AF2 filtering. The de novo extensions of passing designs generally had well-packed ɑ-helical repeat structures.

Extended Data Fig. 5. SEC of extended bifaceted pD5 nanoparticles.

Preparative SEC chromatograms are shown for each of the five extended pD5 nanoparticles shown in Fig. 4. The gray rectangles depict the fractions pooled for further characterization.

Extended Data Fig. 6. Experimental characterization of additional extended bifaceted pD5 nanoparticles.

From top to bottom: Additional assemblies extended by 25, 50, 75, and 100 Å, or extended by 50 Å and rotated by 25°. From left to right: computational design models, preparative SEC chromatograms, raw micrographs of (ABC)₅, and raw micrographs of (ABC)₅-(ABD)₅. Scale bars: 100 nm.

Extended Data Fig. 7. DLS of SEC-purified extended bifaceted pD5 nanoparticles.

DLS and measured hydrodynamic diameters of additional assemblies extended by 25, 50, 75, and 100 Å, or extended by 50 Å and rotated by 25°.

Extended Data Fig. 8. Experimental characterization of pD5-14_rd47.

a, Schematic of pD5-14 ABC heterotrimer redesign to generate pD5-14_rd47 ABC heterotrimers with exterior-facing N termini. The newly diffused ɑ-helix is shown in dark gray, and N and C termini are indicated by blue and red circles, respectively. b, Preparative SEC chromatogram of pD5-14_rd47. c, DLS of SEC-purified pD5-14_rd47 (ABC)₅-(ABD)₅. d, nsEM characterization of pD5-14_rd47. Left: Raw micrographs are shown for the (ABC)₅ and (ABD)₅ components as well as the SEC-purified (ABC)₅-(ABD)₅ assemblies. Right: 2D class averages and a 3D reconstruction of pD5-14_rd47 (ABC)₅-(ABD)₅. Scale bars: 50 nm (raw micrographs) and 20 nm (2D class averages).

Extended Data Fig. 9. Characterization of pD5-14_rd106.

a, Schematic of pD5-14 ABC heterotrimer redesign to generate pD5-14_rd106 ABC heterotrimers with exterior-facing N termini. The newly diffused ɑ-helix is shown in dark gray, and N and C termini are indicated by blue and red circles, respectively. b, Preparative SEC chromatogram of pD5-14_rd106. c, DLS of SEC-purified pD5-14_rd106 (ABC)₅-(ABD)₅. d, nsEM characterization of pD5-14_rd106. Left: Raw micrographs are shown for the (ABC)₅ and (ABD)₅ components as well as the SEC-purified (ABC)₅-(ABD)₅ assemblies. Right: 2D class averages and a 3D reconstruction of pD5-14_rd106 (ABC)₅-(ABD)₅. Scale bars: 50 nm (raw micrographs) and 20 nm (2D class averages).

Extended Data Fig. 10. SEC and nsEM of Neo-2/15–pD5–Neo-2/15 and 41bb_mb1–pD5–41bb_mb1 assemblies.

Top: Preparative SEC chromatograms of Neo-2/15–pD5–Neo-2/15 and 41bb_mb1–pD5-14–41bb-mb1. The fractions collected for further characterization are marked by gray rectangles. Bottom: nsEM raw micrographs of the SEC-purified assemblies. Scale bar: 50 nm for both micrographs.