De novo targeting to the cytoplasmic and luminal side of bacterial microcompartments

Abstract

Bacterial microcompartments, BMCs, are proteinaceous organelles that encase a specific metabolic pathway within a semi-permeable protein shell. Short encapsulation peptides can direct cargo proteins to the lumen of the compartments. However, the fusion of such peptides to non-native proteins does not guarantee encapsulation and often causes aggregation. Here, we report an approach for targeting recombinant proteins to BMCs that utilizes specific de novo coiled-coil protein–protein interactions. Attachment of one coiled-coil module to PduA (a component of the BMC shell) allows targeting of a fluorescent protein fused to a cognate coiled-coil partner. This interaction takes place on the outer surface of the BMC. The redesign of PduA to generate an N-terminus on the luminal side of the BMC results in intact compartments to which proteins can still be targeted via the designed coiled-coil system. This study provides a strategy to display proteins on the surface or within the lumen of the BMCs.

Guiding biomolecular interactions in cells using de novo protein-protein interfaces

“…We find that in most cases the CC behavior mirrors that seen in vitro. Some of the peptide sequences tested here have been shown recently to assemble in E. coli in other contexts: the heterodimeric CC drives the assembly of a novel cytoscaffold and the subcellular localization of active enzymes when fused to shell proteins of a bacterial microcompartment; 6,7 and, while the work presented here was in preparation, the same heterodimeric CCs have been shown by others to recruit T7 RNA polymerase to Zn-finger DNA-binding domains. 28 Some adverse context-dependent effects have been noted: in our activation experiments proximity effects may inhibit heterodimerization; and in the programmable T7 RNA polymerase system the hierarchy of CC interaction strength varies with the nature of the Zn-finger domains to which the peptides are fused.…”

Biotechnological Advances in Bacterial Microcompartment Technology

“…Attachment of the cognate coiled-coil peptide to a fluorescent cargo protein then directed the fluorophore to the outside of the BMC. The redesign of PduA from a BMC-H C to a permuted conformation BMC-H P then allows for the N-terminus to be located on the inside of the compartment, thereby allowing targeting of cargo to the interior [60]. A similar idea of using affinity handles and covalent linkages to target proteins to the lumen of recombinant BMCs has also been reported with the H. ochraceum system [61].…”

“…The same coiled-coil peptide system has also been used to target protein cargo to either the inside or outside of a recombinant BMC in vivo [60]. In this case, one of the coiled-coil peptides was attached to the N-terminus of PduA, which is located on the outer-facing (cytoplasmic) concave side of the protein.…”

The de novo design of α-helical peptides for supramolecular self-assembly

“…Alongside this, a number of de novo α-helical peptides and small proteins have been presented that operate in cells [26]. From the Woolfson basis set, homo-and heterodimer peptides can replace the protein-protein interaction domains in transcription regulation machinery, while CC-Di-AB can direct the intracellular localisation of a synthetic "cytoscaffold", as well as tether enzymes to its surface [68][69][70]. The Baker laboratory has designed specific hydrogen-bonding networks within fourhelix bundles to generate a suite of heterodimers that assemble orthogonally in E. coli [71].…”