Engineering protein nanocages as carriers for biomedical applications

Bhaskar, Sathyamoorthy; Lim, Sierin

hment. Ren et al. showed that the E2 protein can be modified both on the internal and external surfaces for loading of drug molecules inside and for displaying functional epitopes on the outer surface of the nanocage simultaneously. 21 Thus, the E2 protein cage offers promising avenues for tailored engineering of the exterior, the subunit-subunit interfaces and the interior to produce the desired modifications. 22 Vaults. First reported in 1986, vaults are ribonucleoparticles with a mass of 13 MDa found in most eukaryotic cells. 23 Vaults have a barrellike structure measuring 41 × 41 × 72.5 nm 3 with extending caps at their extremities and a hinged waist region. 24 The nanostructure consists of several highly conserved proteins, including the major vault protein (MVP: PDB IDs 2QZV, 4V60), which constitutes over 70% of the overall mass of the nanoparticle, vault poly(ADP-ribose) polymerase (VPARP), telomerase-associated protein 1 (TEP1) and some untranslated RNA molecules. 25 The basic vault structure consists of 78-96 MVPs arranged from the C to N terminus from the cap to the waist, forming a thin protein coat covering an internal cavity of 5 × 10 7 Å 3 . The N terminus is tucked towards the interior of the vault. The MVPs are non-covalently associated. Despite the strong binding between MVPs, the vault has a dynamic structure that opens and closes transiently, a phenomenon referred to as breathing, to incorporate small molecules, proteins and other macromolecules within the inner core. 24 . Peptide extensions at the N termini (shown in red) and C termini (shown in blue) from the waist region (light red) and the extreme ends of the caps, respectively (Aa and b). By fusing an exogenous protein at INT binding site (Ac) and additional peptide at the N terminus (Ad), a composite assembly with multifunctional units could be formed (Ae) 26 Recombinant production of MVPs using a baculovirus expression system in insect cells shows that the protein structure can sufficiently aid in directing the formation of recombinant vault particle with a structure analogous to the wild-type particles. The naturally occurring vault nanocages can thus be engineered for a wide range of novel applications. 28 Other natural scaffolds. Bacterial species have been found to host self-assembling nano-and microsized structures called bacterial microcompartments (BMCs) containing protein complexes formed by 60-20,000 copies of one or more self-assembled protein species. BMCs are part of specific metabolic pathways of the cell, encapsulating unstable or toxic intermediate metabolites. 1 Carboxysomes were the first bacterial organelles identified to perform such functions. As described in 1973, carboxysomes have icosahedral structures with a cross-section measuring 100-150 nm and a protein coat containing 6-10 distinct proteins; they are involved in autotrophic CO 2 fixation in bacteria via RuBisCO and carbonic anhydrase enzymes. 1 Similar to viral capsids, the formation of carboxysomes' icosahedral structure requires a combination of hexameric and pentameric self-assembling units of BMC proteins. Interestingly, the self-assembly property of the carboxysomes was retained in vivo even after the deletion of the RuBisCO enzyme. 29 The flat sheet of the icosahedron is formed by the hexameric units that assemble side by side, whereas the vertices are occupied by the pentameric units ( Figure 5 ). A common BMC subunit consists of 90 amino acids with an α-/β-fold structure. 1 I