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.

Figure 1

(A) Amino acid sequence of insulin-containing B-chain (upper) and A-chain (lower) with disulfide bridges as indicated, (B) insulin monomer (chain A in red and chain B in green; PDB 2JV1), and (C) insulin dimer, showing tyrosine residues as yellow sticks (PDB 2OMI). (D) Insulin hexamer assembly with the Zn ion at the center coordinated to the B10His residue from each monomer unit.

Figure 2

Far-UV CD of (A) free insulin and Zn–insulin and that of (B) insulin–protamine, Zn–insulin–protamine, and protamine. The concentrations of protamine, insulin, and ZnSO₄ were 20, 20, and 10 μM, respectively. (C) % Helical content of insulin and Zn–insulin with and without protamine. Thermal stability curve of (D) free insulin and Zn–insulin and (E) insulin–protamine and Zn–insulin–protamine using ultraviolet–visible (UV–vis) spectrometer. (F) Plot of CD ellipticity at 208 nm vs temperature curve of insulin–protamine and Zn–insulin–protamine. Table showing a comparison of T_m values. Far-UV CD melting spectra of (G) insulin–protamine and (H) Zn–insulin–protamine. Phosphate buffer (10 mM) containing 100 mM NaCl at pH 8.0 was used. The insulin/Zn molar ratio was 1:0.5, and the insulin/phenol molar ratio was 1:3.

Figure 3

ITC thermograms as a result of titration of protamine into insulin at (A) 100 mM and (B) 150 mM salt concentration, and also at different temperatures of (C) 15 °C, (D) 25 °C, and (E) 35 °C. Concentrations were as follows: protamine, 300 μM (1.53 mg mL^–1), and insulin, 60 μM (0.35 mg mL^–1). All of the experiments were performed in 10 mM phosphate buffer at pH 8.0 on a MicroCal iTC200.

Figure 4

ITC thermograms as a result of titration of protamine into Zn–insulin at (A) 100 mM and (B) 150 mM salt concentration and also at different temperatures of (C) 15 °C, (D) 25 °C, and (E) 35 °C. Concentrations were as follows: protamine, 300 μM or 1.53 mg mL^–1; insulin, 40 μM or 0.23 mg mL^–1; ZnSO₄, 20 μM or 0.0035 mg mL^–1; and phenol, 120 μM or 0.011 mg mL^–1. All of the experiments were performed in 10 mM phosphate buffer at pH 8.0.

Figure 5

Plot of ΔH vs temperature for insulin and Zn–insulin showing ΔC_p (slope).

Figure 6

Titration of increasing concentrations of ANS into (A) free insulin, (B) Zn–insulin, (C) Zn–insulin–protamine. The dotted line indicates free ANS excited at 388 nm. All of the experiments were performed in 10 mM phosphate buffer containing 100 mM NaCl at pH 8.0 at 25 °C. The insulin/Zn molar ratio was 1:0.5, and the insulin/phenol molar ratio was 1:3.

Figure 7

Fluorescence titration of increasing concentration of protamine into (A) insulin and (B) Zn–insulin; (C) plot of [(f_o/f – 1)] vs 1/[Q] using the modified Stern–Volmer equation to determine quenching constants. The intensity was recorded at 304 nm. All of the experiments were performed in 10 mM phosphate buffer at pH 8.0 and 25 °C. The insulin/Zn molar ratio was 1:0.5, and the insulin/phenol molar ratio was 1:3.

Figure 8

((A–C) From top to bottom) Topography of (A) insulin, (B) Zn–insulin, and (C) protamine–Zn–insulin assembly, followed by (in the respective row) 3D image of the topography, single-molecule statistical analysis with histogram distributions of cross-sectional height of a respective species. In the last column, it is shown how molecules get arranged at different stages in a pictorial form. Insulin units are represented in gray color (monomer, dimer, or hexamer) and protamine in green.

Figure 9

(A) Insulin dimer interface showing residues (in pink) of chain B (from each monomer) responsible for monomer–monomer interactions (PDB: 4INS). (B) Docking result of insulin (PDB: 2JV1) with protamine (yellow surface). (C) Demonstration of the interactions at the binding interface of insulin and protamine. (D) Hydrophobic interactions between residues of insulin and protamine. (E) Insulin showing exposed hydrophobic residues (sticks) that are not interacting with protamine (yellow surface) and (F) the complex between insulin and receptor (αCT; 704–719 amino acids), showing the crucial residues of insulin involved in receptor binding; gray-shaded area represents the αCT surface (PDB: 4OGA). It can be seen that the hydrophobic residues, which are not involved in the binding in (E) are involved in receptor binding in (F).