Genomic and molecular dynamics analysis of mefA-encoded macrolide efflux protein from Tn2009 and Tn2010 in
Yustinus Maladan1, Endah Retnaningrum2, Budi Setiadi Daryono3
1Eijkman Research Center for Molecular Biology, Cibinong Science Center, The National Research and Innovation Agency, Bogor, 16911, Indonesia. yust013@brin.go.id.
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.
Insights
Antibiotic resistance in Streptococcus pneumoniae to erythromycin is linked to the mefA gene. This study reveals the efflux protein
Area of Science:
- Microbiology
- Structural Biology
- Computational Biology
Background:
- Antibiotic resistance in Streptococcus pneumoniae, especially to macrolides like erythromycin, poses a significant clinical challenge.
- The mefA gene, often found on transposons Tn2009 and Tn2010, encodes a macrolide efflux protein contributing to this resistance.
Purpose of the Study:
- To investigate the genetic characteristics of the mefA gene in Indonesian clinical isolates of Streptococcus pneumoniae.
- To elucidate the mechanism of erythromycin efflux mediated by the mefA gene product using computational approaches.
Main Methods:
- Whole-genome sequencing to identify transposons Tn2009 and Tn2010.
- Multiple sequence alignment to assess mefA gene conservation.
- Homology modeling, molecular docking, and molecular dynamics simulations (200 ns) to study the efflux protein structure and function.
- Steered molecular dynamics and umbrella sampling to analyze the energy landscape of erythromycin translocation.
Main Results:
- High conservation (98-100% identity) of the mefA gene was observed in Indonesian clinical isolates.
- The mefA efflux protein model showed stable binding of erythromycin within its channel.
- Molecular dynamics simulations indicated structural stability of the protein and localized flexibility in a key loop.
- Erythromycin translocation required significant external force, suggesting an active transport mechanism involving multiple binding sites and substantial energy barriers (PMF of ~45 kcal/mol).
Conclusions:
- The study provides detailed structural and energetic insights into mefA-mediated erythromycin resistance in Streptococcus pneumoniae.
- The findings support a resistance mechanism involving stable substrate binding, protein flexibility, and energy-intensive active transport.
- These insights highlight potential targets for developing novel efflux pump inhibitors to combat antibiotic resistance.
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