Characterization of LL37 Binding to Collagen through Peptide Modification with a Collagen-Binding Domain.

Collagen-based biomaterials loaded with antimicrobial peptides (AMPs) present a promising approach for promoting wound healing while providing protection against infections. In our previous work, we modified the AMP LL37 by incorporating a collagen-binding domain (cCBD) as an anchoring unit for collagen-based wound dressings. We demonstrated that cCBD-modified LL37 (cCBD-LL37) exhibited improved retention on collagen after washing with PBS. However, the binding mechanism of cCBD-LL37 to collagen remained to be elucidated. In this study, we found that cCBD-LL37 showed a slightly higher affinity for collagen compared to LL37. Our results indicated that cCBD inhibited cCBD-LL37 binding to collagen but did not fully eliminate the binding. This suggests that cCBD-LL37 binding to collagen may involve more than just one-site-specific binding through the collagen-binding domain, with non-specific interactions also playing a role. Electrostatic studies revealed that both LL37 and cCBD-LL37 interact with collagen via long-range electrostatic forces, initiating low-affinity binding that transitions to close-range or hydrophobic interactions. Circular dichroism analysis showed that cCBD-LL37 exhibited enhanced structural stability compared to LL37 under varying ionic strengths and pH conditions, implying potential improvements in antimicrobial activity. Moreover, we demonstrated that the release of LL37 and cCBD-LL37 into the surrounding medium was influenced by the electrostatic environment, but cCBD could enhance the retention of peptide on collagen scaffolds. Collectively, these results provide important insights into cCBD-modified AMP-binding mechanisms and suggest that the addition of cCBD may enhance peptide structural stability and retention under varying electrostatic conditions.