Formation of Protamine and Zn–Insulin Assembly: Exploring Biophysical Consequences

Abstract

The insulin−protamine interaction is at the core of the mode of action in many insulin formulations (Zn + insulin + protamine) and to treat diabetes, in which protamine is added to the stable form of hexameric insulin (Zn−insulin). However, due to the unavailability of quantitative data and a high-resolution structure, the binding mechanism of the insulin− protamine complex remains unknown. In this study, it was observed that Zn−insulin experiences destabilization as observed by the loss of secondary structure in circular dichroism (CD), and reduction in thermal stability in melting study, upon protamine binding. In isothermal titration calorimetry (ITC), it was found that the interactions were mostly enthalpically driven. This is in line with the positive ΔC m value (+880 cal mol −1 ), indicating the role of hydrophilic interactions in the complex formation, with the exposure of hydrophobic residues to the solvent, which was firmly supported by the 8-anilino-1-naphthalene sulfonate (ANS) binding study. The stoichiometry (N) value in ITC suggests the multiple insulin molecules binding to the protamine chain, which is consistent with the picture of the condensation of insulin in the presence of protamine. Atomic force microscopy (AFM) suggested the formation of a heterogeneous Zn−insulin−protamine complex. In fluorescence, Zn−insulin experiences strong Tyr quenching, suggesting that the location of the protamine-binding site is near Tyr, which is also supported by the molecular docking study. Since Tyr is critical in the stabilization of insulin self-assembly, its interaction with protamine may impair insulin's self-association ability and thermodynamic stability while at the same time promoting its flexible conformation desired for better biological activity.

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

“…An earlier experimental and docking study by Aggarwal et. al . suggested increased flexibility of the complex upon binding, which is contrary to our results.…”

“… is a significant contributor to the binding energy. The fact that values are substantially lower than values indicates that hydrophobic interactions are not as significant as electrostatic interactions, which is consistent with the experimental study done on an equimolar mixture of insulin and protamine . Therein, it was shown that the binding interactions between insulin and protamine are hydrophilic in nature.…”

“…We speculate a higher polarity of P1 in comparison to the other three peptides as revealed by its largest negative electrostatic energy in the bound state with the insulin hexamer. Our study suggests the binding of protamine on the initial half of the B-chain, which may also lead to reduced levels of deamination in the insulin–protamine complex as reported previously . Throughout the MD run, insulin’s hexameric state in the insulin–protamine complex is retained, accounting for its dynamic stability.…”

“…The fact that values are substantially lower than values indicates that hydrophobic interactions are not as significant as electrostatic interactions, which is consistent with the experimental study done on an equimolar mixture of insulin and protamine. 48 Therein, it was shown that the binding interactions between insulin and protamine are hydrophilic in nature. This is also expected as protamine contains arginine amino acids, which mainly interact through H-bonding and electrostatic interactions.…”

“…The four protamine peptides were docked independently to determine the precise binding locations and to understand whether any of the peptides bind to insulin in NPH formulations preferentially over the others. We observed the following in the docked insulin− protamine complexes: (a) insulin's hexameric state is retained upon binding with protamine peptides, (b) protamine peptides bind exclusively on the surface of the insulin R6 hexamer, (c) all four protamine peptides bind to insulin in nearly similar orientation, (d) B-chain residues of insulin are mostly involved in binding with the protamine residues, which is consistent with a previous docking study of protamine peptide with an insulin monomer, 48 and (e) upon binding, protamine peptides retained their random coil structure. It is well known that insulin coexists as a monomer and a dimer, which self- assembles to form an inactive hexameric state, which is key for insulin's storage and prolonged release into the bloodstream.…”