Metal‐dependent assembly of a protein nano‐cage

Abstract

Short, alpha‐helical coiled coils provide a simple, modular method to direct the assembly of proteins into higher order structures. We previously demonstrated that by genetically fusing de novo–designed coiled coils of the appropriate oligomerization state to a natural trimeric protein, we could direct the assembly of this protein into various geometrical cages. Here, we have extended this approach by appending a coiled coil designed to trimerize in response to binding divalent transition metal ions and thereby achieve metal ion‐dependent assembly of a tetrahedral protein cage. Ni2+, Co2+, Cu2+, and Zn2+ ions were evaluated, with Ni2+ proving the most effective at mediating protein assembly. Characterization of the assembled protein indicated that the metal ion–protein complex formed discrete globular structures of the diameter expected for a complex containing 12 copies of the protein monomer. Protein assembly could be reversed by removing metal ions with ethylenediaminetetraacetic acid or under mildly acidic conditions.

A single residue can modulate nanocage assembly in salt dependent ferritin

“…Recently the concept of using metals not as catalysts but to direct protein self-assembly has been developed. 60,61 A number of protein cages held together by such metal-protein interactions, wherein metals are located at well-defined positions, have been demonstrated. 23,62,63 In the case of TmFtn the situation appears more complex compared to designed protein cages: Residue E65 in the wild type protein appears to be strongly repulsed by two other neighbouring negatively charged residues, glutamate 131 and aspartate 134 (Fig.…”

Connectability of protein cages

“…When the trimeric esterase was equipped with a coiled-coil motif designed to trimerize upon binding with divalent metal ions, the dened tetrahedral cage formation was observed by addition of Ni(II), Co(II), Cu(II), and Zn(II). 51 We have shown a simple, distinct strategy for construction of articial protein cages by connecting protein subunits via metal coordination (Fig. 4I).…”

Engineering spatiotemporal organization and dynamics in synthetic cells

“…Substantial advances have been made with regard to computationally designed protein shells and cages (Bale et al, 2016; Brouwer et al, 2019; Cannon et al, 2020; Hsia et al, 2016). In addition, novel design concepts based on symmetry relationships and metal‐mediated assembly have also been achieved (Badieyan et al, 2017; Cannon, Ochoa, & Yeates, 2019; Cristie‐David et al, 2019; Cristie‐David & Marsh, 2019; Malay et al, 2019). So far, these systems have not been used in a synthetic cell context or as functional protein organelles with enzymatic cargo.…”