Isopeptide bonds mechanically stabilize spy0128 in bacterial pili.

Pili on the surface of Streptococcus pyogenes play a crucial role in adhesion to and colonization in human cells. The major pilin subunit, Spy0128, features intramolecular covalent isopeptide bonds that autocatalytically form between the side chains of lysine and asparagine residues and are regarded as important factors in conveying structural stability. In support of this notion, single-molecule force spectroscopy experiments with Spy0128 recently demonstrated the inextensibility of these bonds under mechanical load. However, the molecular determinants of their apparent absolute durability remain unknown. Here, we studied the impact of the isopeptide bond in the Spy0128 C-terminal domain on the mechanical properties of this subunit using force-probe molecular dynamics simulations and force distribution analysis. Even in the presence of the covalent cross-link, the pili β-sandwich domain undergoes partial unfolding, albeit at ∼50% higher rupture forces and with the ability to rapidly refold on the nanosecond timescale. We find that the isopeptide bond is located right at the point of stress concentration in the protein, leading to relative, yet not absolute, mechanical stabilization by the additional cross-link. Our findings indicate how the isopeptide bond enhances the mechanical stability and refolding capability at the molecular level, ensuring that the domain remains predominantly in a potentially adhesive conformation.