Genomic and molecular dynamics analysis of mefA-encoded macrolide efflux protein from Tn2009 and Tn2010 in Streptococcus pneumoniae.

Abstract

Antibiotic resistance in Streptococcus pneumoniae remains a major clinical challenge, particularly for macrolides such as erythromycin. This resistance is commonly associated with transposons Tn2009 and Tn2010 carrying the mefA gene, which encodes a macrolide efflux protein. This study investigated the genetic characteristics of mefA from Indonesian clinical isolates and elucidated its efflux mechanism using integrated genomic analysis and molecular dynamics simulations. Whole-genome sequencing confirmed the presence of Tn2009 and Tn2010 in clinical isolates. Multiple sequence alignment showed high conservation of mefA (98-100% identity), indicating strong evolutionary stability within the species. A homology model of the mefA encoded efflux protein was constructed and used for molecular docking, revealing stable erythromycin binding within the efflux channel with multiple favorable binding poses. Molecular dynamics simulations (200 ns) demonstrated structural stability of the protein, with an average root mean square deviation of 0.174 nm. Root mean square fluctuation analysis identified localized flexibility in residues Asn195-Ser199, suggesting a functionally relevant intracellular loop involved in conformational dynamics. Despite stable interactions, erythromycin remained confined within a localized channel region and did not undergo spontaneous translocation under equilibrium conditions. Steered molecular dynamics simulations indicated that ligand transport requires external force to overcome an initial energetic barrier of 553.07 kJ/mol/nm, followed by stepwise displacement through multiple transient binding sites, consistent with a multi-site relay mechanism. Umbrella sampling further revealed a rugged free energy landscape, with a maximum potential of mean force (PMF) of ~ 45 kcal/mol near the channel exit region. The PMF profile, reconstructed using WHAM across ~ 30 windows, showed well-converged overlap and multiple intermediate minima, indicating a stable sampling of the reaction coordinate. Collectively, these findings provide structural and energetic insights into mefA mediated erythromycin resistance in S. pneumoniae. The results support a mechanism involving stable substrate binding, localized conformational flexibility, and substantial energy barriers requiring active transport, highlighting potential targets for efflux inhibition strategies.