(773f) Free Energy Calculation for Microcin J25 Variants Binding to the Fhua Receptor and to RNA Polymerase

Computer simulations are performed to study the mechanism of the antimicrobial peptide microcin J25 (MJ25), a 21-mer peptide with an unusual lasso structure and high activity against Gram-negative bacteria. MJ25 enters cells by binding to the outer membrane receptor FhuA. Once inside cells MJ25 targets RNA polymerase to inhibit transcription. First, molecular dynamics simulation is implemented to study the binding mechanism of MJ25 on the FhuA receptor and to identify important binding residues (hot spots). The absolute binding free energy calculated from enhanced sampling methods, free energy perturbation (FEP) and thermodynamic integration (TI) agrees with experimental data. In addition, computational mutation analysis reveals that His5 is the key residue responsible for MJ25 and FhuA association. The preservation of the number of hydrogen bonds after mutation is key for MJ25 permeation. Second, data-driven docking is applied to find the binding position of MJ25 on E. Coli RNA polymerase. The protocol for free energy calculation is again implemented to find essential residues, and the results agree with mutagenesis experiment.

These results collectively shed light onto the mechanism of action of MJ25, and offer a roadmap for designing peptide analogues with high activity against Gram-negative bacteria. Such peptides may offer a solution to the significant challenge of bacterial strains emerging that are resistant to all of our antibiotics. Indeed new antimicrobial technologies are urgently needed and although antimicrobial peptides are a promising solution, limited understanding of their mechanism of action at the molecular level has hampered efforts to develop new AMP-based therapeutics.