Versatile selection technology for intracellular protein–protein interactions mediated by a unique bacterial hitchhiker transport mechanism

Abstract

We have developed a reliable genetic selection strategy for isolating interacting proteins based on the ''hitchhiker'' mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway. This method, designated FLI-TRAP (functional ligand-binding identification by Tat-based recognition of associating proteins), is based on the unique ability of the Tat system to efficiently cotranslocate noncovalent complexes of 2 folded polypeptides. In the FLI-TRAP assay, the protein to be screened for interactions is engineered with an Nterminal Tat signal peptide, whereas the known or putative partner protein is fused to mature TEM-1 ␤-lactamase (Bla). Using a series of c-Jun and c-Fos leucine zipper (JunLZ and FosLZ) variants of known affinities, we observed that only those chimeras that expressed well and interacted strongly in the cytoplasm were able to colocalize Bla into the periplasm and confer ␤-lactam antibiotic resistance to cells. Likewise, the assay was able to efficiently detect interactions between intracellular single-chain Fv (scFv) antibodies and their cognate antigens. The utility of FLI-TRAP was then demonstrated through random library selections of amino acid substitutions that restored (i) heterodimerization to a noninteracting FosLZ variant, and (ii) antigen binding to a low-affinity scFv antibody. Because Tat substrates must be correctly folded before transport, FLI-TRAP favors the identification of soluble, nonaggregating, protease-resistant protein pairs and, thus, provides a powerful tool for routine selection of interacting partners (e.g., antibody-antigen), without the need for purification or immobilization of the binding target.ligand binding proteins ͉ protein folding quality control ͉ signal peptide ͉ twin-arginine translocation ͉ 2-hybrid system P rotein-protein interactions are key molecular events that integrate multiple gene products into functional complexes in virtually every cellular process. Because such interactions mediate numerous disease states and biological mechanisms underlying the pathogenesis of bacterial and viral infections, identification of protein-protein interactions remains one of the most important challenges in the postgenomics era. The yeast 2-hybrid (Y2H) system (1) has been the tool of choice for revealing numerous protein-protein interactions, underlying diverse protein networks and complex protein machinery inside living cells. To date, Y2H has been used to generate protein interaction maps for humans (2), Drosophila melanogaster (3), Caenorhabditis elegans (4), Saccharomyces cerivisiae (5, 6), vaccinia virus (7), and Escherichia coli bacteriophage T7 (8). Another important application of the Y2H methodology is the discovery of diagnostic and therapeutic proteins, whose mode of action is high-affinity binding to a target peptide or protein. For example, several groups have isolated antibody fragments that are readily expressed in the cytoplasm of cells where they bind specifically to a desired target (9, 10), and in certain instances ablate protein functi...

Split‐Cre recombinase effectively monitors protein–protein interactions in living bacteria

“…All but two of the clones (96%) contained at least one Leu in either the second or third d position of the bZIP domain heptad repeat (Table 1). This strong enrichment agrees with previous findings showing that bZIP pairs [30][31][32][33], including Jun and Fos specifically [27][28][29], are largely insensitive to single point mutations in the d position of the heptad repeat. As expected, the clone corresponding to a full reversion back to the canonical Leu/Leu residues was isolated most often, with a frequency of 29%.…”

“…To determine if reduced affinity between Jun-Fos domains could be discriminated with our assay, we mutated the second leucine (Leu2Val, L2V) or the second and third leucines (L2V, L3V) in the bZIP domain of FosLZ. Previous work has shown that mutations such as these to the d position in the bZIP domain heptad repeat reduce affinity between the JunFos pair in the order (highest to lowest affinity): FosLZ > FosLZ(L2V) >> FosLZ(L2/3V) [27][28][29]. In agreement with these earlier studies, cells co-expressing Jun-LZ with the FosLZ(L2V) mutant were slightly less fluorescent (MF = 2066) than the JunLZ-FosLZ pair.…”

“…It is worth mentioning that we previously investigated the amino acid preferences in the second d position of FosLZ using a different protein-protein interaction assay in bacteria. Isolated clones contained Leu, Ala, Phe, Arg, and His in the same rank order as found here except for Gln, which ranked above Phe in that study [29]. Based on these consistencies, we conclude that our split-Cre PCA is a reliable new method for phenotypic enrichment of competent binders expressed from combinatorial protein libraries.…”

“…Two clones were isolated that did not contain Leu at either position (Met/Phe and Ala/Trp). While not the strongest clones in terms of fluorescence, these clones contain residues that are functional at general d positions in leucine zippers [30] and have been isolated in previous mutational studies [29]. These residues have high stability in helical formation [34] and are suitable for hydrophobic burial given their nonpolar chemistry.…”

Ubiquibodies, Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing

“…These results also confirm the potential of CHIP for customizable target degradation. Indeed, given the plethora of existing DBPs against known cellular targets and the availability of robust technologies for on-demand isolation of new DBPs that function inside cells (26,36,37), ubiquibodies are likely to become a powerful tool for reverse genetics. In addition to the wide array of endogenous proteins that can be targeted with newly designed ubiquibodies, the existing R4-uAb construct described here could be used in conjunction with ␤-gal protein trapping (38) to silence virtually any ␤-galtagged protein expressed from its endogenous loci in cultured cells and whole organisms.…”