Abstract
Gram-positive bacteria can resist large mechanical perturbations during their invasion and colonization by secreting various surface proteins with intramolecular isopeptide or ester bonds. Compared to isopeptide bonds, ester bonds are prone to hydrolysis. It remains elusive whether ester bonds can completely block mechanical extension similarly to isopeptide bonds, or whether ester bonds dissipate mechanical energy by bond rupture. Here, we show that an ester-bond containing stalk domain of Cpe0147 is inextensible even at forces > 2 nN. The ester bond locks the structure to a partially unfolded conformation, in which the ester bond remains largely water inaccessible. This allows the ester bond to withstand considerable mechanical forces and in turn prevent complete protein unfolding. However, the protecting effect might be reduced at non-physiological basic pHs or low calcium concentrations due to destabilizing the protein structures. Inspired by this design principle, we engineer a disulfide mutant resistant to mechanical unfolding under reducing conditions.
Highlights
Gram-positive bacteria can resist large mechanical perturbations during their invasion and colonization by secreting various surface proteins with intramolecular isopeptide or ester bonds
These results suggest that the interplay between the ester bond and the protein structure is critical to the mechanical properties of the C1 domain
By employing single-molecule force spectroscopy, protein engineering, and molecular dynamics simulation, we studied the nanomechanical properties of the C1 domain of Cpe0147 from Clostridium perfringens with a spontaneously formed intramolecular ester bond
Summary
Gram-positive bacteria can resist large mechanical perturbations during their invasion and colonization by secreting various surface proteins with intramolecular isopeptide or ester bonds. Recent studies revealed that the reactivity of thioester bonds is high at low forces and can be completely blocked at forces larger than 35 pN, entailing the bacteria stress dependent mobility[24] Ester bond is another type of intramolecular covalent bonds discovered in Gram-positive surface proteins and is formed between the side chains of Thr and Gln residues[9,16,25]. Molecular dynamics simulations reveal that the ester bond locks the structure to a partially unfolded conformation, in which the ester bond remains largely water inaccessible This allows the ester bond to withstand considerable mechanical forces and in turn to prevent complete protein unfolding. The environment-dependent mechanical stability may be related to the biological functions of Cpe0147 in vivo
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
