Binding free energy analysis of colicin D, E3 and E8 to their respective cognate immunity proteins using computational simulations

Authors: KOIRALA, MAHESH - Orise Fellow; Fagerquist, Clifton

Submitted to: Molecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/7/2025
Publication Date: 3/12/2025
Citation: Koirala, M., Fagerquist, C.K. 2025. Binding free energy analysis of colicin D, E3 and E8 to their respective cognate immunity proteins using computational simulations. Molecules. 30(6). Article 1277. https://doi.org/10.3390/molecules30061277.
DOI: https://doi.org/10.3390/molecules30061277

Interpretive Summary: Colicins (Col) are natural protein antibiotics (whose genes are encoded in plasmids) utilized by bacteria to attack bacterial neighbors as a survival strategy especially in challenging environments where limited resources can result in intense bacterial competition. Colicin-bearing plasmids (small circular pieces of DNA) have been shown to increase the fitness, survivability and persistence of bacterial foodborne pathogens. Plasmids are transferred between bacteria by horizontal gene transfer resulting in the spread of virulence genes critical to pathogen survival. Co-expressed with the plasmid-encoded colicin gene (col) is an immunity gene (im) whose protein binds to and temporarily inactivates the colicin protein thus suppressing its attack on the bacterial host that produced it. The binding of the Col-Im complex is believed to be highly specific. In order to better understand the function of Col-Im, we used artificial intelligence (i.e. Alphafold2) to determine the structure of three Col-Im complexes that are frequently carried by plasmid-bearing environmental isolates of pathogenic E. coli collected from an agricultural region in California. The Col-Im structures obtained were then analyzed using molecular dynamic (MD) simulations that probed complex stability over time followed by calculations of the binding energies of these complexes. It was found that van der Waals and electrostatic interactions as well as hydrogen bonding played major roles in the binding of these complexes. Knowledge of these intermolecular interactions should allow design of synthetic colicin variants that have no natural immunity cognate, and, in consequence, a pathogen would have no natural defense against an attack by such a colicin variant.

Technical Abstract: Colicins are antimicrobial proteins produced by bacteria for the purpose of destroying neighboring bacteria. Colicin activity is neutralized by a specific cognate immunity protein in order to protect the host. This study investigates the structural and binding mechanisms underlying the interaction of colicin-D, -E3 and -E8 to their respective immunity proteins (ImD, Im3 and Im8) using structure prediction, molecular dynamics (MD) simulations and MM-PBSA approach of free energy calculations. High-confidence colicin-immunity (Col-Im) complex structures predicted using AlphaFold2 were subjected to MD simulations of 150 ns with GROMACS and were analyzed for the binding free energy calculation using gmx_MMPBSA. Results showed that the complex of Col_E3-Im3 exhibited the most favorable binding free energy, driven by strong van der Waals and electrostatic interactions. Col_D-ImD and Col_E8-Im8 also showed the favorable binding. Electrostatics and hydrogen bonding emerged as a key factor driving binding and stability, while polar solvation acted as a destabilizing factor across all systems. These outcomes provide an understanding of the molecular mechanisms of Col-Im systems, with potential applications for developing natural antimicrobials for food safety.