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

Abstract

PqsE and RhlR, key regulators of the Pseudomonas aeruginosa quorum sensing (QS) system, form a hetero-tetrameric 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 °C) compared to mammalian body temperature (37 °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 °C and 37 °C. The results demonstrate that the overall stability of the complex decreases at 37 °C, with global RMSD analysis indicating greater fluctuations compared to 25 °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 °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 °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.