Connectability of protein cages

Abstract

Regular, hollow proteinaceous nanoparticles are widespread in nature. The well-defined structures as well as diverse functions of naturally existing protein cages have inspired the development of new nanoarchitectures with desired capabilities. In such approaches, a key functionality is "connectability". Engineering of interfaces between cage building blocks to modulate intra-cage connectability leads to protein cages with new morphologies and assembly-disassembly properties. Modification of protein cage surfaces to control inter-cage connectability enables their arrangement into lattice-like nanomaterials. Here, we review the current progress in control of intra- and inter-cage connectability for protein cage-based nanotechnology development.

Fig. 1. Protein cage comparison. (I and II) Cryo-EM structures of the prolate head of bacteriophage T4 (EMD: 6323) (I) and the procapsid shell of bacteriophage P22 (EMD: 5149) (II). (III and IV) Crystal structures of human heavy chain ferritin (PDB: 2FHA) (III) and Aquifex aeolicus lumazine synthase (PDB: 1HQK) (IV). Note the difference in the scale bars, 100 Å for (I), and (II) and 10 Å for (III) and (IV).

Fig. 2. Connectability in natural and re-engineered protein capsids. (I) Hydrophobic interactions stabilizing the bacteriophage P22 capsid. (a) Two neighbouring capsomers of P22. (b) Enlarged image of the region surrounded by the box in (a) shows the residues forming the hydrophobic peg and pocket. Highlighted residues were subjected to mutagenesis. Reproduced with permission from ref. 19; Copyright (2019) American Society for Microbiology. (II) Bacteriophage HK97 capsomer cross-linking. (a) Three neighbouring subunits are covalently linked by isopeptide bonds (highlighted in the enlarged view). (b) Electron density map of the isopeptide bond formed by the ε amino group of the Lys169 of one subunit and the γ carbon of the Asn356 amide group of the other subunit. Reproduced with permission from ref. 23; Copyright (2000) The American Association for the Advancement of Science. (III) Change in the structure of the native human heavy chain ferritin (HFtn) subunit after deletion of six amino acids residues. Native HFtn subunit (b) with highlighted deleted amino acids (yellow) and two parts of the helix, which are rearranged after deletion. When the C-terminal part of the helix (purple) moves toward the N-terminal part (cyan), a subunit termed H_α is produced (a). The opposite rearrangement generates a subunit termed H_β (c). Reproduced with permission from ref. 25; Copyright (2016) American Chemical Society. (IV) Regulation of the ferritin quaternary states by introducing/deleting disulphide bonds. (a) Four of each subunit, H_α (blue) and H_β (purple), assemble into 8-mer bowl-like proteins (NF-8). (b) Deletion of intra S–S bond results in the conversion of NF-8 into a 24-mer ferritin-like protein cage (red). (c) Insertion of inter S–S bonds led to the conversion of NF-8 into a 16-mer protein cage. (d) Deletion of the same intra S–S and insertion of the inter S–S bonds caused the conversion of NF-8 into a 48-mer protein cage. Reproduced with permission from ref. 26; Copyright (2019) Springer Nature. (V) Circular permutation of Aquifex aeolicus lumazine synthase (AaLS). (a) Structure of the wild type AaLS (AaLS-wt) pentamer (left). One monomer unit is coloured: residues 1–119, orange; residues 120–156, blue. The native termini in AaLS-wt are connected by peptide linker with different lengths ((L_xH_y), where x and y represent the number of total amino acids and histidines, respectively) and new termini are introduced between residues 119 and 120, resulting in circularly permuted AaLS (cpAaLS) (right). (b) The assemblies of cpAaLS variants possessing different linkers form structures depicted on negative stain TEM images (scale bar = 100 nm). Reproduced from ref. 30 with permission from The Royal Society of Chemistry.

Fig. 3. Strategies for artificial protein cage formation through protein–protein interactions. (I) Symmetry-based design and computationally re-engineered interfaces. After choosing initial architecture (here tetrahedral) with 3-fold symmetry axes (a) and arrangement of four copies each of two different trimeric proteins (green and blue) along symmetry axes (b and c), potential interfaces with multiple contacting amino-acids were remodelled using computational simulations (d and e). Reproduced with permission from ref. 31; Copyright (2014) Springer Nature. (II) Multimeric fusion proteins as building blocks. Dimeric and trimeric proteins, shown by the green and red shapes, respectively (a), are fused via semi-rigid linker (b), resulting in self-assembly into artificial cage (c). Reproduced with permission from ref. 36; Copyright (2001) National Academy of Sciences, U.S.A. (III) Coiled coils with different oligomerization states fused with trimeric esterase enable formation of cages with planned geometry. Red dot indicates esterase C-termini and a point of fusion. Reproduced with permission from ref. 42; Copyright (2017) Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (IV) Self-assembled cage-like particles (SAGEs). Helixes from homotrimeric (green) and heterodimeric (red-blue) coiled coils are linked via disulphide bonds to form six-helix building blocks. When mixed, building blocks assemble into a hexagonal network. Reproduced with permission from ref. 45; Copyright (2013) American Association for the Advancement of Science. (V) Analogy between DNA and protein folds. (a and b) Native structures of a DNA aptamer (a) and a globular protein (b) can be formed by complex interactions between residues. (c and d) Modular folds (origami) of DNA (c) and polypeptide (d) are both stabilized by simple and defined pairing systems. Reproduced with permission from ref. 47; Copyright (2014) Wiley Periodicals, Inc.

Fig. 4. Strategies for artificial protein cage formation through metal coordination. (I) Au(i)-mediated TRAP-cage assembly. (a) TRAP ring structure in two orthogonal views with substituted Cys35 shown as red spheres (left), and the compound used as the Au(i) source (right). (b) Model of TRAP-cage overlapped with its electron density map. Gold atoms are shown as spheres. (c) Magnified image at the TRAP ring–ring interface. The arrowheads indicate density bridges connecting neighbouring rings. Reproduced with permission from ref. 52; Copyright (2019) Springer Nature. (II) Components and structure of bimetallic cages. (a) Structure of cytochrome cb₅₆₂. Residues constituting metal-binding sites are shown as sticks. (b) Zn(ii) and Fe(iii)-binding motifs inducing protein dimerization and trimerization, respectively. (c) Structure of the dodecameric bimetallic cage with enlarged Zn(ii) and Fe(iii)-binding motifs. Fe(iii) and Zn(ii) ions are represented as orange/red and blue spheres, respectively. Reproduced with permission from ref. 55; Copyright (2020) Springer Nature.

Fig. 5. Artificial protein cages as a tool for macromolecular display. (I and II) Structures of two-component artificial cages genetically modified with envelope protein-derived antigens from RSV (DS-Cav1) (I) or HIV-1 (BG505 SOSIP) (II). The scaffold assembly consists of 20 trimeric (I53-50A) and 12 pentameric (I53-50B) building blocks, and antigens are fused with the trimeric subunits. Reproduced with permission from ref. 58 and 59; Copyright (2019) Elsevier and Springer Nature. (III) Structural model of protein scaffold for cryo-EM imaging. The artificial protein cage with tetrahedral symmetry is composed of 12 copies each of two protein subunits, A (yellow) and B (orange). DARPin (green) fused with subunit A via helical linker contains binding surface (pink) to capture cargo protein (blue) for cryo-EM imaging. Reproduced with permission from ref. 60; Copyright (2019) Springer Nature.

Fig. 6. Assembling protein cages into lattices. (I) Common strategy for lattice assembly utilizing electrostatic interactions, where protein cage surface charges (e.g. TmFtn) are countered by complimentarily charged connectors (e.g. positively charged AuNPs) to drive their assembly. Reproduced with permission from ref. 66; Copyright (2019) American Chemical Society (DOI: 10.1021/acs.nanolett.9b01148, further permissions related to the material excerpted should be directed to the ACS). (II) An octacationic zinc phthalocyanine 1 and a tetraanionic pyrene 2 derivative used as a connector for construction of the ferritin lattice affords light-induced singlet oxygen production. Reproduced with permission from ref. 18; Copyright (2015) American Chemical Society (DOI: 10.1021/acsnano.5b07167, further permissions related to the material excerpted should be directed to the ACS). (III) Positively charged dendrimer serves as a template for lattice formation of P22 particles fused with negatively charged peptide. 3D structure is further locked with cementing protein, followed by template removal. Reproduced with permission from ref. 67; Copyright (2018) American Chemical Society. (IV) Engineered HFtn with positive (Ftn(pos)) and negative (Ftn(neg)) surface charge self-assemble into a tetragonal lattice. Reproduced with permission from ref. 69; Copyright (2016) American Chemical Society. (V) Lattice assembly utilizing aromatic stacking. Substitution to phenylalanine (top) or tyrosine (bottom) on the exterior surface of HFtn results in formation of a 2D array or 3D lattice, respectively. Reproduced with permission from ref. 70; Copyright (2018) American Chemical Society. (VI) Metal coordination-assisted lattice formation. Selection of suitable protein nodes equipped with metal-binding motifs, metal ions with preferred geometries, and dihydroxamate linkers with different shapes and lengths results in formation of cubic or tetragonal lattices. Reproduced with permission from ref. 71; Copyright (2017) American Chemical Society. (VII) Scheme of crack formation and self-healing of the Ca-HFtn crystal-hydrogel network (left). Light microscopy images of crystal–hydrogel hybrids, depicting the self-healing of Ca-induced cracking (scale bare = 100 μm) (right). Reproduced with permission from ref. 72; Copyright (2018) Springer Nature.