Rational disruption of the oligomerization of the mini‐ferritin E. coli DPS through protein‐protein interface mutation

Abstract

DNA-binding protein from starved cells (DPS), a mini-ferritin capable of self-assembling into a 12-meric nano-cage, was chosen as the basis for an alanine-shaving mutagenesis study to investigate the importance of key amino acid residues, located at symmetry-related protein-protein interfaces, in controlling protein stability and self-assembly. Nine mutants were designed through simple inspection, synthesized, and subjected to transmission electron microscopy, circular dichroism, size exclusion chromatography, and ''virtual alanine scanning'' computational analysis. The data indicate that many of these residues may be hot spot residues. Most remarkably, two residues, R83 and R133, were observed to shift the oligomerization state to~50% dimer. Based on the hypothesis that these two residues constitute a ''hot strip,'' located at the ferritin-like threefold axis, the double mutant was generated which completely shuts down detectable formation of 12-mer in solution, favoring a cooperatively folded dimer. The fact that this effect logically builds upon the single mutants emphasizes that complex self-assembly has the potential to be manipulated rationally. This study should have an impact on the fundamental understanding of the assembly of DPS protein cages specifically and protein quaternary structure in general. In addition, as there is much interest in applying these and similar systems to the templation of nano-materials and drug delivery, the ability to control this ferritin's oligomerization state and stability could prove especially valuable.

Dps biomineralizing proteins: multifunctional architects of nature

“…This interface between two monomers along the two-fold axis of the four-helix bundle is also architecturally maintained in ferritin and Bfr molecules and may resemble the initial assembly driving the entire assembly process [1,40]. Although the stability of Dps complexes is reported to be high, point mutations in the vicinity of the trimer interface can significantly destabilize the Dps complex from E. coli, leading to the disassembly of the complex into dimers [134].…”

Designability of Aromatic Interaction Networks at E. coli Bacterioferritin B-Type Channels

“…Consistent with the SEC, N132W forms cages. In addition, we have noted in the past that some protein cage mutants that are crippled in their ability to form the cage state in solution generate TEM-observable cages [ 24 ]. We attributed this observation to the evaporated and surface-immobilized conditions required for TEM, which may be acting to force unfavorable cages together and this is the case with N34W which forms only dimers in solution but cages in TEM conditions.…”

The Oligomeric Form of the Escherichia coli Dps Protein Depends on the Availability of Iron Ions

“…However, of the two amino acid substitutions (W52A and D78A) made at this protein interface near the ferroxidase centers, only W52A slightly decreased the stability of Dps [ 30 ]. On the other hand, the substitution R133A located on the opposite site of the monomer bundle (indicated in violet in Figure 3 B) and positioned near the ferritin-like three-fold symmetry axes shifted the oligomerization state to ~50% of dimers [ 30 ]. The docking performed for the entire Dps globule revealed Fe 2 O 3 oxides near these residues ( Figure 3 B).…”