CCCtag – a standardized designed set of coiled-coil heterodimerizing modules for tunable regulation of localization, transcriptional activation and light- and chemical signal based regulation

Protein-protein interactions are involved in almost all cellular processes, they define scaffolds, regulate cellular localization and participate in functioning in cellular machines, such as those performing transcription, replication, splicing and others. In order to manipulate cellular processes we need a reliable standardized set of orthogonal protein-protein interaction modules. 

Coiled-coil peptide heterodimers are small polypeptide modules whose specificity is defined by a combination of electrostatic and hydrophobic interactions. They have been used to design a new type of protein folds, coiled-coil protein origami, where the structure is defined by pairs of CC modules that self-assemble into polyhedra unknown in nature within a single polypeptide chain (Gradišar et al., 2013; Ljubetič et al., 2017). Screening of proteome interactions of de novo modules designed by the Jerala group (NICP set) suggested good orthogonality in mammalian cells, therefore we decided to investigated the use those modules as tools to regulate mammalian cells. Experimental analysis of pairwise interactions of 12 CC peptides confirmed very high orthogonality in human cells with typically three orders of magnitude preference for the correct pairs over the background. Moreover the affinity of each CC pair could be tuned by modifying residues at noncontact sites and by combining several peptides, to tune the stability within the 100-fold range. Orthogonality of three CC pairs was demonstrated by their use as cellular localization tags, defined by CC-pairing specificity. Coiled coil modules can be used to tune transcriptional regulation by concatenation of selected number of repeats where the concatenated CC peptide tag (CCC-tag) could amplify the transcription of selected reporter or genomic gene targets based on CRISPR/dCas9/gRNA guiding and achieved the superior potency in comparison to several previously designed strategies, such as SunTag and SamTag. Moreover, the similar approach was used to amplify the response of the chemically- or light-regulated transcriptional activation, which strongly increased the response in mammalian cells to a chemical stimulus as well as in cells implanted into the animals.

The NICP set and its implementations therefore represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biology and other uses in molecular biology. A particular advantage of this platform is that it has quite small footprint, allows define the recruitment of one or several molecular partners in a selected stoichiometry and minimal interactions with other cellular components.

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Text by: Roman Jerala, Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia

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