Diversification of Protein Cage Structure Using Circularly Permuted Subunits

Abstract

Self-assembling protein cages are useful as nanoscale molecular containers for diverse applications in biotechnology and medicine. To expand the utility of such systems, there is considerable interest in customizing the structures of natural cage-forming proteins and designing new ones. Here we report that a circularly permuted variant of lumazine synthase, a cage-forming enzyme from Aquifex aeolicus (AaLS) affords versatile building blocks for the construction of nanocompartments that can be easily produced, tailored, and diversified. The topologically altered protein, cpAaLS, self-assembles into spherical and tubular cage structures with morphologies that can be controlled by the length of the linker connecting the native termini. Moreover, cpAaLS proteins integrate into wild-type and other engineered AaLS assemblies by coproduction in Escherichia coli to form patchwork cages. This coassembly strategy enables encapsulation of guest proteins in the lumen, modification of the exterior through genetic fusion, and tuning of the size and electrostatics of the compartments. This addition to the family of AaLS cages broadens the scope of this system for further applications and highlights the utility of circular permutation as a potentially general strategy for tailoring the properties of cage-forming proteins.

Laboratory evolution of virus-like nucleocapsids from nonviral protein cages

“…S1). Unlike another recently reported AaLS circular permutant with a different ring-opening site (18), the resulting protein, cpAaLS(84), forms homogeneous spherical structures similar in size to WT AaLS assemblies (SI Appendix, Fig. S2A).…”

“…To enable RNA binding, the cationic λN+ peptide, engineered from the λ-phage antitermination complex, was appended to the new N terminus to give λcpAaLS ( Fig. 1A) (18,19). The 5′-and 3′-UTRs of the corresponding gene were equipped with artificial BoxBr packaging signals, consisting of a BoxB stem-loop structure that binds λN+ peptides (19), plus a fluorogenic Broccoli aptamer (20) for detection purposes (SI Appendix, Fig.…”

Enzyme encapsulation by protein cages

“…A patchwork assembly method addresses this problem by mixing cargo-fused monomer with unmodied monomer in different ratios to generate cages with different numbers of cargo in different protein cages, such as CCMV and AaLS. 65,66 There are several instances of protein cages known where cargo encapsulation is carried out using genetic fusion. 67 Among these perhaps the most notable is the P22 bacteriophage cage VLP where protein cargo encapsulation has almost exclusively been achieved using genetic fusion.…”

Substrate Sorting by a Supercharged Nanoreactor

“…In principle, leakage of the protease from the cage could be prevented by covalently linking the enzyme to the lumenal surface, for example, by modifying circularly permuted capsid subunits. 25 …”