The insight into the intermolecular interactions between protamine and insulin lispro

Area of Science:

• Pharmacology and Biochemistry
• Protein-Ligand Interactions
• Drug Delivery Systems

Background:

• Protamine forms complexes with Insulin Lispro (IL) to extend its hypoglycemic effect.
• The precise binding mechanism between protamine and IL is not fully understood.
• Understanding these interactions is crucial for optimizing insulin formulations.

Purpose of the Study:

• To elucidate the binding mechanism between protamine components and Insulin Lispro (IL).
• To identify which protamine peptide exhibits the strongest binding affinity with IL.
• To explore the structural and energetic contributions to protamine-IL complex formation.

Main Methods:

• Reversed-phase high-performance liquid chromatography (RP-HPLC) to assess binding capacity.
• Isothermal titration calorimetry (ITC) to determine binding thermodynamics.
• Surface plasmon resonance (SPR) to analyze binding kinetics.
• Bioinformatics analysis and molecular dynamics (MD) simulations to investigate interaction details.

Main Results:

• Binding capacity varied among protamine peptides, with Peptide 2 (P2) showing the highest affinity for IL.
• ITC revealed an enthalpy-driven binding mode for P2 with IL (ΔH = -149 ± 2.24 kJ/mol).
• SPR indicated rapid association and slow dissociation kinetics for protamine-IL complexes.
• Bioinformatics and MD simulations highlighted the importance of hydrogen bonds and electrostatic interactions, with distinct differences between peptides.

Conclusions:

• Peptide 2 demonstrates the strongest binding affinity to Insulin Lispro, suggesting its potential for enhanced insulin formulations.
• The binding is primarily enthalpy-driven, involving significant hydrogen bonding and electrostatic interactions.
• This multi-perspective study provides a deeper mechanistic understanding of protamine-IL interactions, supporting regional binding models.