Abstract
The overall stability of globular protein structures is marginal, a balance between large numbers of stabilizing non-covalent interactions and a destabilizing entropic term. Higher stability can be engineered by introduction of disulfide bonds, provided the redox environment is controlled. The discovery of stabilizing isopeptide bond crosslinks, formed spontaneously between lysine and asparagine (or aspartic acid) side chains in certain bacterial cell-surface proteins suggests that such bonds could be introduced by protein engineering as an alternative protein stabilization strategy. We report the first example of an isopeptide bond engineered de novo into an immunoglobulin-like protein, the minor pilin FctB from Streptococcus pyogenes. Four mutations were sufficient; lysine, asparagine and glutamic acid residues were introduced for the bond-forming reaction, with a fourth Val/Phe mutation to help steer the lysine side chain into position. The spontaneously-formed isopeptide bond was confirmed by mass spectrometry and X-ray crystallography, and was shown to increase the thermal stability by 10 °C compared with 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.
Highlights
The adhesin FbaB24, both from Streptococcus pyogenes
The isopeptide bond in Spy0128-N is formed between Lys-36 on its first β-strand and Asn-168 on its last β-strand, with the spontaneous intramolecular reaction being catalyzed by Glu-117 on the fifth β-strand[1]
The domain is split into two parts, a receptor domain lacking its final β-strand that carries the key Asn/Asp residue, and a peptide that contains the sequence of the missing strand
Summary
The adhesin FbaB24, both from Streptococcus pyogenes. Spontaneous isopeptide bond formation has been demonstrated to occur when these domains are split into a peptide carrying the Asn/Asp residue and a receptor with the complementary Lys and Glu residues. The host protein is FctB, the basal subunit in S. pyogenes pili[27] This single-domain protein has the same CnaB-type fold as is found for many isopeptide-containing domains, but has none of the required Lys, Glu and Asn residues at the usual crosslinking site. It has substantial structural differences, with only 15% sequence identity and a root-mean-square difference (rmsd) of 1.8 Å over 96 Cαpositions when compared with its closest homolog, the N-terminal domain of Spy012827. The stability of the domain is substantially enhanced, raising the possibility of engineering crosslinks of this kind into other Ig-like domains as the “rules” governing bond formation become better understood
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