Modulation of Coiled-Coil Dimer Stability through Surface Residues while Preserving Pairing Specificity

Drobnak, Igor; Gradišar, Helena; Ljubetič, Ajasja; Merljak, Estera; Jerala, Roman

doi:10.1021/jacs.7b01690

Cited by 64 publications

(78 citation statements)

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“…We recently showed that the stability of CC dimers can be modified without affecting pairing specificity by varying surface residues 20 . As high stability of CC dimer modules might lead to folding bottlenecks, we sought to decrease their stability by introducing acidic residues at positions b, c, and f of the CC heptad repeats (Fig.…”

Section: Resultsmentioning

confidence: 99%

Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo

et al. 2017

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Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized >20 single-chain protein cages in three shapes-tetrahedron, four-sided pyramid, and triangular prism-with the largest containing >700 amino-acid residues and measuring 11 nm in diameter. Their stability and folding kinetics were similar to those of natural proteins. Solution small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophysical analysis confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.

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Section: Resultsmentioning

confidence: 99%

Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo

et al. 2017

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“…Split SEA fragments were fused with coiled-coil dimer-forming P3 and P4 polypeptides ( Fig. 2A) designed and tested previously in our lab (19,20), that form a tight and specific heterodimer. Both polypeptide domains were genetically fused with SEA fragments by a 10 amino acid glycine-serine peptide linker.…”

Section: Screening Methods For the Detection Of Effective Split Sea Dementioning

confidence: 99%

Design of split superantigen fusion proteins for cancer immunotherapy

Golob-Urbanc

Rajčević

Strmšek

et al. 2019

Journal of Biological Chemistry

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Edited by Peter CresswellSeveral antibody-targeting cancer immunotherapies have been developed based on T cell activation at the target cells. One of the most potent activators of T cells are bacterial superantigens, which bind to major histocompatibility complex class II on antigen-presenting cells and activate T cells through T cell receptor. Strong T cell activation is also one of the main weaknesses of this strategy as it may lead to systemic T cell activation. To overcome the limitation of conventional antibody-superantigen fusion proteins, we have split a superantigen into two fragments, individually inactive, until both fragments came into close proximity and reassembled into a biologically active form capable of activating T cell response.A screening method based on fusion between SEA and coiledcoil heterodimers was developed that enabled detection of functional split SEA designs. The split SEA design that demonstrated efficacy in fusion with coiled-coil dimer forming polypeptides was fused to a single chain antibody specific for tumor antigen CD20. This design selectively activated T cells by split SEA-scFv fusion binding to target cells.Cancer therapy is experiencing great progress, especially based on application of biological drugs and cell immunotherapy. Nevertheless, cancer remains one of the leading causes of death in developed countries. In the last decade, immunotherapy became an important approach for fighting cancer, where the main goal is to activate the patient's own immune system to specifically recognize and kill tumor cells. Antibody-based therapeutics that target surface antigens expressed on tumor cells are successfully used for treatment of different types of cancer. Although unconjugated monoclonal antibodies are efficient, clinical studies showed that conjugating cytotoxic agents to monoclonal antibodies enhances their clinical utility (1). Antibody-drug conjugates are a class of highly potent biological drugs, composed of an antibody and an effector molecule. One of the immunotherapy approaches to potentiate the effects of monoclonal antibodies includes superantigens as effector molecules.

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“…Ligand‐mediated assembly of CC dimers would be therefore of considerable interest for various biotechnological applications such as sensing, encapsulation and drug delivery. Here we present the design of a CC dimer‐based conformational switch the assembly of which is mediated by metal ions as well as by pH (Scheme ), and is furthermore orthogonal to the existing heterodimeric CC set used for the construction of coiled‐coil protein origami (CCPO) cages …”

Section: Methodsmentioning

confidence: 99%

SwitCCh: Metal‐Site Design for Controlling the Assembly of a Coiled‐Coil Homodimer

2018

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Conformational change of proteins in response to chemical or physical signals is the underlying principle of many regulatory and transport mechanisms in biological systems. The ability to design proteins the conformational state of which can be precisely and reversibly controlled would facilitate the development of molecular machines tailored for specific applications. Here we explore metal‐binding site design to engineer a peptide‐based conformational switch called SwitCCh that assembles into a homodimeric coiled‐coil in response to the addition of ZnII ions or low pH. Addition of ZnII promoted formation of a parallel homodimer with an increase in thermal stability by more than 30 °C. The peptide could be reversibly cycled between the coiled‐coil and random conformation. Furthermore, the SwitCCh peptide was orthogonal to the previously developed coiled‐coil dimer set, indicating it could be used for regulated self‐assembly of coiled‐coil based nanostructures and materials.

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Modulation of Coiled-Coil Dimer Stability through Surface Residues while Preserving Pairing Specificity

Cited by 64 publications

References 50 publications

Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo

Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo

Design of split superantigen fusion proteins for cancer immunotherapy

SwitCCh: Metal‐Site Design for Controlling the Assembly of a Coiled‐Coil Homodimer

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