Molecular dynamics simulations decipher the structure–function relationship of a key bacterial amyloid peptide and its cytotoxic mutants. Experimentally, a Lys17Ala mutation reduces α-helicity and cytotoxicity, while an Asp13Ala mutation reduces helicity but paradoxically enhances toxic activity. To elucidate the underlying atomistic mechanisms, we performed extensive sampling for the wild-type (WT) peptide and both single-point mutants (Ala13, Ala17). Our computational strategy included twenty independent classical MD simulations (4 µs per variant) for robust sampling, supplemented by 400 ns of well-tempered metadynamics per variant to explore free energy landscapes and metastable states exhaustively.

Time-structured Independent Component Analysis (TICA) identified the dominant conformational states, confirming the experimental helical trend: WT > Ala17 > Ala13. Our results reveal that residue 17 is critical for C-terminal helix stabilization via specific intramolecular contacts. Conversely, the Ala13 mutation disrupts a key salt bridge, resulting in a pronounced increase in N-terminal flexibility. We propose that this enhanced conformational freedom and an altered amphipathic profile may promote deeper insertion into and more effective disruption of cell membranes, explaining the elevated cytotoxicity despite lower helicity.

This study provides the crucial mechanistic basis for the mutants' divergent behaviors, highlighting that while one residue acts as a primary structural stabilizer, the other serves as an electrostatic regulator. These detailed insights are crucial for understanding bacterial amyloid toxicity and can guide the rational development of novel anti-staphylococcal therapies.