Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization

Abstract

An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic β-cell. Due to the transient nature of insulin dimer, direct investigation of this important form is inherently difficult. To address the relationship between insulin oligomerization and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further oligomerization to form the structural equivalent of the classical hexamer. The covalently linked dimer neither bound to the insulin receptor, nor induced a metabolic response in vitro. However, it was extremely thermodynamically stable and did not form amyloid fibrils when subjected to mechanical stress, underlining the importance of oligomerization for insulin stability.

Figure 1. Cartoon representation of the crystal structure of the B25C-dimer.

A: The A chain is coloured in green and the B chain is shown in blue. The additional disulphide bond is shown by stick representation (yellow). An omit map was calculated by omitting the Sulphur atom of B25C. The resulting difference electron density Fo-Fc map is coloured in orange at σ-level = 3.0. It is clear from the structure that the two monomers are linked by a disulfide bond between the two adjoining B25C. B: Comparison of the B25C structure (blue) with that of the porcine in-sulin (PDB code 1B2E) (grey). The Cα trace shows that the two structures have a high resemblance with minor deviations in Cα positions at residue B21E and B29K.

Figure 2. AUC results for the B25C-dimer.

A: SV Analysis of the B25C-dimer in the presence of 2 Zn²⁺/hexamer (insulin normals). In the top part of the figure, open circles represent the g(s*)/s-curve derived from a dcdt-analysis. For clarity, only every 10^th data point is shown. The solid red line represents the fit to a model of a single ideal species, resulting in the parameters shown in Tabel 2. The bottom part of the figure represents the local deviations between the experimental and simulated data (residuals). Every data point is shown. The rmsd of the shown fit is 9.83×10⁻³. B: Representative data of a SE experiment used to determine the self-association model of B25C. In the top part of the figure, open circles represent experimental concentration distributions at apparent thermo- and hydrodynamic equilibrium for one concentration (out of five) at 15 krpm (black), 24 krpm (red) and 36 krpm (green). For clarity, only every 10^th data point is shown. The solid like-colored lines represent the global fit to all measured conditions to a model of a reversible monomer-dimer model, resulting in the equilibrium coefficient mentioned in the text. The bottom part of the figure represents the local deviations between the experimental and simulated data (residuals). Every data point is shown. The molar mass parameter was fixed to its expected value and the global rmsd of the fit is 7.4×10⁻³.

Figure 3. Measurements of in vitro activity of the B25C-dimer compared to HI.

A: Representative insulin receptor binding curves for HI(black), B25C-NEM1 (dark gray) B25C-NEM2(gray)and the B25C dimer(light gray). B: Representative metabolic dose response curves for HI(black) and the B25C-dimer (dark gray). Each point on the graph represents the mean ± SD, n = 4 within one assay.

Figure 4. Assessing the stability of the B25C-dimer compared to HI.

A: DSC of HI and the B25C-dimer. B: ThT fibrillation assay of 0.3 mM B25C-dimer (grey diamonds) and 0.6 mM HI (black diamonds) with incubation at 37°C and vigorous shaking as described in “Methods”. Both samples contained 7 mM phosphate adjusted to pH 7.4.