Unraveling the molecular mechanism of temperature-induced destabilization in the PqsE-RhlR complex of Pseudomonas aeruginosa at mammalian body temperature through classical molecular dynamics and metadynamics

PqsE and RhlR, key regulators of the Pseudomonas aeruginosa quorum sensing (QS) system, form a heterotetrameric complex essential for controlling the expression of virulence factors such as pyocyanin. The interaction between the PqsE homodimer and the RhlR homodimer bound to C4-HSL, enables RhlR to bind low- affinity promoters, thereby influencing gene regulation. Recent studies suggest that RhlR transcriptional activity is modulated by temperature, exhibiting higher activity at environmental temperatures (25 degrees C) compared to mammalian body temperature (37 degrees C). However, the molecular mechanisms underlying this temperature- dependent regulation remain unclear. This study aims to explore how temperature influences the structural stability of the PqsE/RhlR/C4-HSL complex using molecular dynamics (MD) simulations at 25 degrees C and 37 degrees C. The results demonstrate that the overall stability of the complex decreases at 37 degrees C, with global RMSD analysis indicating greater fluctuations compared to 25 degrees C. Further RMSD analysis of PqsE and RhlR separately revealed that the destabilization is more pronounced in RhlR, particularly in its DNA-binding domain (DBD), where significant flexibility and destabilization were observed at 37 degrees C, as indicated by the higher RMSF values. Free energy landscape analysis confirmed increased conformational flexibility in the RhlR at higher temperatures, potentially impairing its DNA-binding ability. To further investigate this, metadynamics simulations were performed for PqsE/RhlR/C4-HSL bound to DNA, revealing a remarkable increase in the distance between RhlR and DNA at 37 degrees C, potentially leading to a faster separation. These findings indicate that temperature-induced destabilization of RhlR, especially in the DBD, may explain the reduced RhlR transcriptional activity observed at mammalian body temperature.