Integrative in vitro and in silico evaluation of HSP60-derived peptides as immunomodulators of the TLR4/MD-2 complex

This study investigated the activity of HSP60-derived peptides on the human and murine TLR4/MD-2 complex, combining in vitro and in silico strategies. TLR4/MD-2 is crucial for recognizing lipopolysaccharide (LPS) and initiating innate immune activation. Although this complex is structurally conserved across species, species-specific interactions were identified that influence peptide-induced activity. Six 15-amino-acid-long peptides were tested in human and mouse endothelial cells. Based on its bioactivity, three different biological patterns were observed: TLR4 overexpression, cytokine production, or no effect. Molecular docking revealed that peptide binding energy alone did not predict bioactivity. Instead, TLR4/MD-2 species-specific interactions played a key role. In mice, peptides 4 and 5 engaged residues associated with LPS activation. Peptide orientation, stable conformation, and acidic residue positioning influenced activation potential. This study highlights the relevance of using multi-conformational docking to uncover subtle but critical interaction patterns. These findings emphasize the need for species-specific analysis in peptide immunotherapy development.