Probing the Interaction of Protamine with Zn-insulin through Biophysical
and Molecular Docking Studies
Abstract
There is a successful use of protamine-insulin formulation
(Zn+insulin+protamine) to treat diabetes in which protamine is added to
the stable form of hexameric insulin (Zn-insulin). The biophysical
characterization of Zn-insulin, which can dissociate to form
biologically active monomers, is well reported. However, its interaction
with protamine, which is at the core of the mode of action in many
pharmaceutical formulations, is unresolved. Through biophysical
characterization, we have tried to dissect the interactions driving the
Zn-insulin-protamine complexation. Based on the thermal melting study,
it was found that protamine indulges in the destabilization of
Zn-insulin. Fluorescence results revealed that Zn-insulin experiences
Tyr quenching in the presence of protamine, undergoing a significant
conformational change. As shown by the molecular docking study,
protamine disturbs the H-bonding network at the dimer interface by
binding to the amino acid residues involved in the dimer stabilization.
It may result in the freeying of B-chain, introducing conformational
fluctuations in the insulin. This is well supported by the loss of
helical content seen in circular dichroism. Further, the
insulin-protamine complex formation was strongly dominated by hydrogen
bonding and a few hydrophobic contacts. The endothermic heat and
positive entropy observed in isothermal titration calorimetry in the
dissociation of Zn-insulin-protamine is a reflection of that. Finally,
the ANS binding study proposed the adaption of a flexible conformation
by the Zn-insulin-protamine complex containing exposed hydrophobic
residues, a potential arrangement for successful receptor binding.