Delving into Macrolide Binding Affinities and Associated Structural Modulations in Erythromycin Esterase C: Insights into the Venus Flytrap Mechanism.

Since their inception in antibacterial therapy, macrolide-based antibiotics have significantly shaped the evolutionary pathways of pathogenic bacteria, driving them to develop diverse antimicrobial resistance (AMR) mechanisms. Among these, macrolide esterase, commonly referred to as erythromycin esterase, emerged as a critical defense mechanism, enabling bacteria to detoxify macrolides by hydrolyzing the macrolactone ring within the bacterial cell. In this study, we delve into the intricate interactions and conformational dynamics of erythromycin esterase C (EreC), a key member of the Ere enzyme family. We have focused on three FDA-approved and widely prescribed macrolides─erythromycin, clarithromycin, and azithromycin─by employing classical molecular dynamics, absolute binding free energy calculations, and 2D well-tempered metadynamics simulations to explore their interactions with EreC. To estimate the absolute binding free energies, we have used the recently developed and robust "Streamlined Alchemical Free Energy Perturbation (SAFEP)" protocol. The results from our molecular dynamics simulations and advanced analyses portrayed the crucial role of hydrophobic interactions within the macrolide binding cleft of EreC, along with the significant influence of the minor lobe in facilitating overall structural fluctuation. In silico alanine scanning identified top three hydrophobic residues, i.e., PHE248, MET333, and PHE344, responsible for macrolide binding inside that cleft. According to the free energy calculations, azithromycin and clarithromycin showed greater binding affinities toward EreC than the parent macrolide erythromycin. Moreover, 2D metadynamics simulations along with graph theory-based eigenvector centrality analyses revealed a metastable "semiopen" state during the hypothesized "active loop closure" of the EreC protein triggered by subtle conformational changes of an important histidine residue, HIS289, upon macrolide capture, drawing a fascinating parallel to the renowned "Venus flytrap" mechanism.