The insight into the intermolecular interactions between protamine and insulin lispro

Protamine, a mixture of polypeptides, can form complexes with Insulin Lispro (IL) to prolong its hypoglycemic effect, but the binding mechanism remains unclear. The four protamine components were used to study their binding mechanism with IL through RP-HPLC, ITC, SPR and bioinformatics analysis. The results of RP-HPLC indicated that the binding capacity of protamine and its four peptides with IL are not constant. As the concentration of protamine increases, the binding amount keeps increasing, with peptide 2 (P2) exhibiting the highest binding capacity among the four peptides. After forming complexes, the structure of IL changed. ITC results showed that among the four components, P2 has the lowest K_D value, with a ΔH of -149 ± 2.24 kJ/mol and -TΔS of 111 kJ/mol, indicating an enthalpy-driven binding mode. SPR results revealed a trend of rapid association and slow dissociation for all components. Bioinformatics analysis, showed that hydrogen bond and electrostatic interaction played an important role in binding, and there was a significant difference between 6 and 12 residues. MD simulations of P1 and P2 showed significant differences in RMSF values. Overall, P2 binds IL most strongly, while P1 has the weakest affinity. This study provides a novel multi-perspective exploration of protamine-IL interactions, with SPR and RP-HPLC supporting regional binding models and enhancing our understanding of their mechanisms.