Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures. agging with peptides (e.g., HA, myc, FLAG, His 6 ) is one of the most common ways to detect, purify, or immobilize proteins (1-4). Peptides are very useful minimally disruptive probes (5) but they are also "slippery"-antibodies or other proteins typically bind peptides with low affinity and poor mechanical strength (6-9). We sought to form a rapid covalent bond to a peptide tag without the use of chemical modification, artificial amino acids, or cysteines (disulfide bond formation is reversible and restricted to particular cellular locations).It has recently been found that Streptococcus pyogenes, like many other Gram-positive bacteria, contains extracellular proteins stabilized by spontaneous intramolecular isopeptide bonds (10). Here we explored the second immunoglobulin-like collagen adhesin domain (CnaB2) from the fibronectin binding protein FbaB, found in invasive strains of S. pyogenes (11,12) and essential for phagocytosis-like uptake of the bacteria by endothelial cells (13). CnaB2 contains a single isopeptide bond conferring exceptional stability: CnaB2 remains folded even at pH 2 or up to 100°C (12). By splitting CnaB2 into peptide and protein fragments, followed by rational modification of the parts, we developed a peptide tag of 13 amino acids that rapidly formed a covalent bond with its protein partner (138 amino acids, 15 kDa) and characterized the conditions for reaction, cellular specificity of bond formation, and resilience of the reacted product.

Peptide tagging is a key strategy for observing and isolating proteins. However, the interactions of proteins with peptides are nearly all rapidly reversible. Proteins tagged with the peptide SpyTag form an irreversible covalent bond to the SpyCatcher protein via a spontaneous isopeptide linkage, thereby offering a genetically-encoded way to create peptide interactions that resist force and harsh conditions. Here, we determined the crystal structure of the reconstituted covalent complex of SpyTag and SpyCatcher at 2.1 Å resolution. The structure showed the expected reformation of the β-sandwich domain seen in the parental streptococcal adhesin, but flanking sequences at both the N- and C-termini of SpyCatcher were disordered. In addition, only 10 out of 13 amino acids of the SpyTag peptide were observed to interact with SpyCatcher, pointing to specific contacts important for rapid split protein reconstitution. Based on these structural insights, we expressed a range of SpyCatcher variants and identified a minimized SpyCatcher, 32 residues shorter, that maintained rapid reaction with SpyTag. Together, these results give insight into split protein β-strand complementation and enhance a distinct approach to ultra-stable molecular interaction.

Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein-peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide-peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide-peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture.bionanotechnology | synthetic biology | metastasis | antibody | nanobiotechnology

SpyTag is a peptide which spontaneously forms an amide bond to its protein partner SpyCatcher. Here we fused SpyTag at the N-terminus of β-lactamase and SpyCatcher at the C-terminus, so the partners could cyclize to lock together the termini of the enzyme. Wild-type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized β-lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher-cyclization achieved > 60 °C increase in stability, much larger than from point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding calorimetrically and via mutants, cyclization did not increase the unfolding temperature of β-lactamase, but facilitated refolding after thermal stress. SpyTag and SpyCatcher sandwiching represents a simple and efficient route for enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.

SpyTag is a peptide which spontaneously forms an amide bond to its protein partner SpyCatcher. Here we fused SpyTag at the N-terminus of β-lactamase and SpyCatcher at the C-terminus, so the partners could cyclize to lock together the termini of the enzyme. Wild-type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized β-lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher-cyclization achieved > 60 °C increase in stability, much larger than from point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding calorimetrically and via mutants, cyclization did not increase the unfolding temperature of β-lactamase, but facilitated refolding after thermal stress. SpyTag and SpyCatcher sandwiching represents a simple and efficient route for enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.

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