Conformational plasticity and truncational effects on bovine lactoferricin: structural determinants of enhanced...

The rapid emergence of multidrug-resistant bacterial pathogens has created an urgent demand for alternative antimicrobial agents. Bovine lactoferricin (Lfcin B), a cationic antimicrobial peptide derived from bovine lactoferrin, exhibits potent broad-spectrum antibacterial activity. Although truncated derivatives of Lfcin B retain partial antimicrobial effects, their efficacy relative to full-length Lfcin B and the structural mechanisms governing their function remain poorly understood.

Full-length Lfcin B and three truncated variants (Lfcin B15, Lfcin B9, and Lfcin B6) were synthesized using solid-phase peptide synthesis and characterized by RP-HPLC and MALDI-TOF-MS. Circular dichroism spectroscopy was employed to evaluate secondary structural changes under different ionic and hydrophobic environments. Tertiary structures were predicted using AlphaFold3. Antibacterial activities were assessed against multidrug-resistant Gram-negative and Gram-positive pathogens, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella gallinarum, Shigella flexneri, Staphylococcus aureus, and Trueperella pyogenes, using MIC, MBC, and agar diffusion assays.

Circular dichroism spectroscopy revealed that ionic strength and hydrophobic environments modulate the secondary structures of the peptides, with increased ionic strength consistently reducing random coil ratios across all variants. Structural stability progressively diminished with peptide truncation, as shorter variants exhibited less conformational complexity. AlphaFold3-predicted tertiary structures identified two distinct conformations for full-length Lfcin B, an α-helix-rich state and a β-sheet-dominant topology, whereas truncated variants adopted simpler structural ensembles, primarily α-helical or random coil conformations. Antibacterial activity decreased markedly with peptide truncation. Lfcin B demonstrated the strongest and broadest-spectrum antibacterial activity, showing substantially lower MIC and MBC values and larger inhibition zones than truncated peptides. In contrast, Lfcin B6 exhibited only limited activity against T. pyogenes. Structural analyses indicated that the intact sequence and intramolecular disulfide bond of Lfcin B are essential for maintaining conformational stability, membrane interaction capacity, and antibacterial potency.

The antibacterial efficacy of bovine Lfcin B is strongly associated with its full-length sequence, conformational adaptability, and disulfide bond-mediated structural stability. Progressive truncation compromises structural plasticity and significantly attenuates antimicrobial activity. These findings support Lfcin B as a promising structural scaffold for the development of next-generation therapeutics against antibiotic-resistant bacterial infections.