SH3, PDZ) and their cognate peptide ligands are frequently used as scaffolds to create programmable, higher-order protein assemblies. , interactions between well-known and widespread adaptor domains (e.g. As for instance nicely demonstrated by a metabolic engineering approach from Dueber et al. Non-covalent linkages are mainly stabilized by hydrophobic and ionic interactions as well as hydrogen bonds between specific protein interaction domains. In living systems-eukaryotes as well as prokaryotes-covalent and non-covalent linkages between and within polypeptides are highly relevant to control protein activity as well as stability and help to organize and regulate metabolic flux or signal transduction pathways. To avoid the risk of dissociation and to allow formation of stable protein complexes, there is a need to develop a toolbox of specific and covalent protein connectors. Furthermore, linkage of several proteins into one functional complex is often limited by the restricted number of comparably efficient and specific interaction domains. However, the design of post-translational multiprotein assemblies is often hampered by weak and reversible protein-protein or protein-peptide interactions. These protein architectures offer a wide range of applications, including for instance efficiency enhancement of metabolic pathways by creating multi-enzyme complexes that facilitate substrate channeling. In synthetic biology and biotechnology, artificial linkage of proteins or peptides plays a crucial role in the formation of stable and complex protein architectures. Our study suggests that splitting into tag and catcher moieties is tolerated by a significant portion of the naturally occurring CnaB-domains, thus providing a large reservoir for the design of novel tag/catcher systems. For these two systems length and sequence variations of the peptide tags were investigated revealing only a relatively small effect on the efficiency of the reaction. Experimental testing for intermolecular isopeptide bond formation demonstrated two of the four systems to be functional. To address this point, we have selected a set of four CnaB domains of low sequence similarity and characterized the resulting tag/catcher systems by computational and experimental methods. However, it is unclear whether domain splitting is generally tolerated within the CnaB-family or only by a small subset of these domains. Two of the presently available tag/catcher systems were derived from closely related CnaB-domains of Streptococcus pyogenes and Streptococcus dysgalactiae proteins. Additional covalent tag/catcher systems would allow creating more complex and ultra-stable protein architectures and networks. There are already numerous biotechnological and medical applications that demonstrate the usefulness of covalent linkages mediated by these systems. Tags and catchers are generated by splitting protein domains that contain intramolecular isopeptide or ester bonds that form autocatalytically under physiological conditions. A promising approach relies on so-called tag/catcher systems that are fused to the proteins of interest and allow a durable linkage via covalent intermolecular bonds. Building proteins into larger, post-translational assemblies in a defined and stable way is still a challenging task.
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