The art of designed coiled-coils for the regulation of mammalian cells

Abstract

Synthetic biology aims to engineer complex biological systems using modular elements, with coiled-coil (CC) dimer-forming modules are emerging as highly useful building blocks in the regulation of protein assemblies and biological processes. Those small modules facilitate highly specific and orthogonal protein-protein interactions, offering versatility for the regulation of diverse biological functions. Additionally, their design rules enable precise control and tunability over these interactions, which are crucial for specific applications. Recent advancements showcase their potential for use in innovative therapeutic interventions and biomedical applications. In this review, we discuss the potential of CCs, exploring their diverse applications in mammalian cells, such as synthetic biological circuit design, transcriptional and allosteric regulation, cellular assemblies, chimeric antigen receptor (CAR) T cell regulation, and genome editing and their role in advancing the understanding and regulation of cellular processes.

Graphical abstract

Figure 1

Design of coiled-coil (CC) peptides (A) Schematic and helical wheel representation of parallel (top) and antiparallel (bottom) CC heterodimers. Positions of amino acid residues in heptad repeats are labeled (a,b,c,d,e,f,g). Hydrophobic (cyan) and electrostatic (magenta) interactions between residues are shown in dashed lnes. (B) Structure of a parallel CC heterodimer. Residues involved in electrostatic and hydrophobic interactions are colored magenta and cyan, respectively. Heptad repeat residues are shown. (C) Representative matrix of a designed set of CC heterodimers. Dark squares indicate the expected interaction between designed heterodimers. (D) Schematic representation of different sets of designed CC peptides (S, NICP, and N) that differ in interaction affinities and thermal stabilities (weak to high).

Figure 2

Protein interaction devices based on coiled coils (A) Two-component transmission systems based on protein-protein interactions. (B) CC-based cell compartment localization. CC dimers were used to direct subcellular protein localization to the plasma membrane, cell nucleus, or cytosol. Cargo delivery into cells and subcellular targeting were also successfully demonstrated by the implementation of cell-penetrating peptides and CC-mediated liposome fusion. (C) Building high-order cellular organizations using CC dimers. A platform for engineering synthetic cell adhesion molecules, termed helixCAM, implemented various CC dimers to transmembrane domains. This enabled the formation of multicellular aggregates of selected mammalian cells. (D) CC dimers-based intercellular communication. A small ubiquitin-like modified (SUMO) tag was fused to a CC dimer, which was secreted by mammalian cells (sender population). Upon binding to the complementary CC peptides on the receiver population a receptor was activated.

Figure 3

Coiled-coil-based engineering of transcriptional regulation and modification of antibodies (A) Split synthetic transcription factor-based system composed of DNA-binding domains (TALE or dCas9) and effector domains, both fused to CC pairs. CC peptides enable the reconstitution of DNA binding and activation domain into an active transcription factor. (B) Recruitment of an exonuclease to the Cas9/gRNA complex via CC interaction for increased gene editing efficiency. (C) Prime edit (PE) facilitated by CC. Virus-like particles (VLPs) were used to deliver a double-stranded break-free gene editing system into cells. (D) SUPRA CAR system, composed of a zipCAR and zipFv, for targeting tumor cells. Fused to each of the two components, CC (leucine zipper) served for reconstitution into a functional receptor. (E) Engineered antibodies that can be selectively activated in the tumor microenvironment. Heterodimeric CC domains served as a steric hindrance on complementarity-determining regions that were exposed only upon action of tumor-associated proteases.

Figure 4

Coiled coils as allosteric modulators of protein function (A and B) The principle of INSRTR allosteric regulation is based on the insertion of a CC-forming peptide into a target site of a protein, allowing the protein to retain its function. This setup allows the design of either (A) OFF-INSRTR, where the binding of a regulatory peptide disturbs protein function or (B) ON-INSRTR, where regulatory peptide releases the autoinhibitory loop, allowing the protein to regain its function. (C) 3D structure of OFF (top) and ON (bottom) INSRTR-TEV protease. The active site is marked in magenta.

Figure 5

Modular protease- and coiled-coil-based molecular circuits (A) Designed molecular circuits that can sense different input signals, process information, and trigger complex cellular responses. (B) Split-protease-cleavable orthogonal-CC-based (SPOC). Strategically positioned protease cleavage sites and CCs enabled different levels of binding and competition. This allowed the creation of a range of two-input Boolean logic circuits. (C) Logic and circuits of hacked orthogonal modular proteases (CHOMP). Similar to SPOCK, CHOMP relies on protease-induced regulation; however, an additional type of regulation is added through the implementation of degrons. (D) Engineered inducible secretion of proteins from the endoplasmic reticulum (ER), lumER, RELEASE, and membER. CC-operated split protease system applied to an inducible protein secretion system enables regulated secretion of therapeutic proteins by an array of inputs. (E) CC-based two-part transmembrane synthetic receptor termed DocTAR. One part carries a protease and the other contains a protease-responsive split transcription factor fused to an autoinhibited antiparallel CC. Ligand-based bridging enables the release of a split transcription factor that is reconstituted in the cytosol by a third part of the system via CC interaction. (F) CC modified proteolytically engineered activators of calcium channels (PACE). The system is composed of a CAD protein domain, which can activate Orai Ca²⁺ channels, and a CC pair that renders the domain inactive. Activation was achieved by strategically incorporating protease cleavage sites positioned at the end of the CC inhibitory region.