Engineered Design of the E-Helix Structure on Ferritin Nanoparticles

Abstract

Insertion of an immunogenic epitope at the C-terminus of ferritin has shown the potential to produce a stable and efficacious vaccine. There is however limited understanding of how C-terminus insertion affects ferritin protein stability. The E-helix at the C-terminus has attracted interest because there are contradictory reports as to whether it has a role in protein stabilization. Here, we report, for the first time, combining molecular dynamics simulation (MDS) with experiment to engineer the design of the E-helix at the C-terminus of engineered human ferritin heavy chain (F 1 ) inserted with Epstein−Barr nuclear antigen 1 (EBNA1, E 1 ) and flexible linker (L 3 ) residues (to afford F 1 L 3 E 1 ). Hot spots on the E-helix of the C-terminus were predicted by MDS at aa 167 (Glu) and aa 171 (Asp). Five (5) variants of F 1 L 3 E 1 were constructed by considering hot spots and alteration of electrostatic or hydrophobic interfaces, namely, (1) C1, hot spots substituted with noncharged residue Gln; (2) C2, hot spots substituted with positively charged residue Arg; (3) C3, hydrophobic residues substituted with the most hydrophobic residues Val and Ile; (4) C4, hydrophobic residues substituted with the most hydrophilic residues Gln and Asn; and (5) C5, a heptad repeat structure in the E-helix disrupted by substituting "a" and "d" heptad residues with noncharged polar residue Gln. It was found that the E-helix is essential to maintain integrated protein stability and that changing the hydrophobic interface (C3 and C4) had more significant effects on protein folding and stability than changing the electrostatic interface (C1 and C2). It was confirmed by both MDS and experiment that variants C1, C2, and C5 were able to fold to form stable conformational structures with protein surface hydrophobicity similar to that of F 1 L 3 E 1 . However, they are less thermally stable than F 1 L 3 E 1 . Significant changes in hydrophobicity drove significant protein aggregation for variants C3 and C4. It is concluded that the molecular design of the C-terminus in engineered ferritin, especially the E-helix, is important to ensure the epitope-based chimeric vaccine is safe (aggregate free) and efficacious.

Engineered human H-chain ferritin with reversed charge of the internal cavity exhibits RNA-mediated spongelike effect for loading RNA/DNA-binding molecules

“…24 The E-helix has been previously utilized as a site for the insertion of an immunogenic epitope (Epstein–Barr nuclear antigen 1) without compromising the folding properties of ferritin. 25 Therefore, in this work, we replaced the six terminal predominantly negatively charged amino acids with a positively charged RKRK amino acid cluster. We were surprised to find that during the self-assembly process in E. coli , the engineered HsaFtH-RK is capable of loading bacterial RNA.…”