Computational Approach to Elucidating Insulin-Protamine Binding Interactions and Dynamics in Insulin NPH Formulations.

Insulin NPH is an intermediate-acting insulin. Its protracted action profile is due to the formation of microcrystalline suspensions when insulin is complexed with a basic peptide protamine, zinc ion, and phenolic ligands. Despite advancements in analytical techniques, the binding epitope and binding mode of the protamine in the insulin-protamine complex are still unknown. In this study, we used bioinformatics tools such as molecular docking and molecular dynamics (MD) simulations to compute the binding sites and energetics of the insulin-protamine complex. We have taken four naturally occurring protamine peptides that are independently docked with the insulin R6 hexamer and subjected them to 200 ns MD simulations to observe the dynamics of the complexes and estimate the binding energies. The arginine-rich protamine peptides were found to bind on the surface of the insulin hexamer through hydrogen bonding, hydrophobic, and electrostatic interactions well supported by the calculated negative binding energies. The overall structure of the insulin hexamer was retained upon binding, highlighting its dynamic stability in the complex. Furthermore, the residues at the termini of the protamine peptides in the complex were seen to be highly dynamic, which stabilize toward the end of the simulation.