Abstract:
Gram-positive bacterial pili and fimbriae are exposed to a hostile environment and must endure high tensile forces during the adhesion process. These ultra-stable proteins often contain internal crosslinks that impart crucial mechanical strength. Here, we report the first example of an isopeptide bond engineered de novo into an immunoglobulin-like protein domain. Through the site-directed mutagenesis of four residues we introduced isopeptide-forming residues into the hydrophobic core of FctB, an IgG-like minor pilin protein from Streptococcus pyogenes, to produce a covalent bond not present in the wild type protein. The isopeptide bond was engineered to covalently link the first and last β-strands, a position that is subjected to the highest mechanical stress. The presence of the engineered isopeptide-containing protein was confirmed by mass spectrometry and X-ray crystallography, and its thermal stability was measured as 81°C, an increase of 10°C over the wild type protein. This novel method for increasing the stability of IgG-like proteins has potential to be adopted by the field of antibody engineering, which share similar β-clasp Ig-type domains.