Computational and structural evidence for neurotransmitter-mediated modulation of the oligomeric states of human insulin in storage granules

Abstract

Human insulin is a pivotal protein hormone controlling metabolism, growth, and aging and whose malfunctioning underlies diabetes, some cancers, and neurodegeneration. Despite its central position in human physiology, the in vivo oligomeric state and conformation of insulin in its storage granules in the pancreas are not known. In contrast, many in vitro structures of hexamers of this hormone are available and fall into three conformational states: T₆, T₃R^f₃, and R₆ As there is strong evidence for accumulation of neurotransmitters, such as serotonin and dopamine, in insulin storage granules in pancreatic β-cells, we probed by molecular dynamics (MD) and protein crystallography (PC) if these endogenous ligands affect and stabilize insulin oligomers. Parallel studies independently converged on the observation that serotonin binds well within the insulin hexamer (site I), stabilizing it in the T₃R₃ conformation. Both methods indicated serotonin binding on the hexamer surface (site III) as well. MD, but not PC, indicated that dopamine was also a good site III ligand. Some of the PC studies also included arginine, which may be abundant in insulin granules upon processing of pro-insulin, and stable T₃R₃ hexamers loaded with both serotonin and arginine were obtained. The MD and PC results were supported further by in solution spectroscopic studies with R-state-specific chromophore. Our results indicate that the T₃R₃ oligomer is a plausible insulin pancreatic storage form, resulting from its complex interplay with neurotransmitters, and pro-insulin processing products. These findings may have implications for clinical insulin formulations.

Keywords: crystal structure; dopamine; insulin; pancreatic islet; serotonin; vesicles.

Figure 1.

General structural organization of the three main forms of insulin hexamer: T₆ (A), T₃R^f₃ (B), and R₆ (C) in the hexamer top view along its 3-fold symmetry axis. Insulin B chains are in white, A chains are in pink; the chains of one representative insulin dimer are indicated in A in light blue (B-chains) and yellow (A-chains). Some N and C termini of one dimer are also indicated in A and only B-chain N termini in B and C, with * corresponding to the symmetry-related monomer within a dimer. The B1-B8 segments of the B chains that contribute to the largest structural changes in TR transitions are in red (top of the hexamer) and in green (bottom of the hexamer). The Zn²⁺ ion is in blue. D–F, representative insulin monomers in T, R^f, and R-state, respectively, with a coloring code as in A–C. Typical, main ligand binding sites I and III in the R insulin form are indicated in C as S1 and S3 respectively.

Figure 2.

Starting conformations of phenol (A, insulin R₆ hexamer shown for the sake of clarity of the phenolic pocket location), serotonin (B), and dopamine (B) molecules (depicted by van der Waals spheres) in the phenolic site I pocket (amino acids A6 and A11 involved in the binding are depicted by a stick model); two initial orientations of serotonin, and dopamine were considered.

Figure 3.

r.m.s.d. of the protein backbone (heavy atoms) from the insulin R₆ hexamer NMR structure with different phenolic ligands (phenol, dopamine, or serotonin). Labels 1 and 2 correspond to the two starting orientations of the neurotransmitter molecules.

Figure 4.

r.m.s.d. from simulations of all phenolic ligands (serotonin or dopamine in two orientations) from the phenol-binding pockets in the insulin R₆ hexamers (in total six phenolic ligands per one R₆ hexamer). Each line corresponds to the r.m.s.d. of one phenolic ligand from its starting position with respect to the O atom of Cys^A6 and the N atom of Cys^A11 involved in a hydrogen bond. A zero or small value of r.m.s.d. means a strong ligand, R₆ hexamer hydrogen bond, whereas a significant increase of r.m.s.d. indicates breaking of this bond.

Figure 5.

Dopamine (top, red) and serotonin (bottom, magenta) spatial distributions around the insulin R₆ hexamer (top view). The A/B-chains are in blue and yellow, respectively; the same isodensity value (∼50× the bulk concentration) was used for both neurotransmitters. Black circles depict the site III binding pockets.

Figure 6.

Serotonin (top) and dopamine (bottom) binding site III formed by two adjacent insulin monomers (Chain A/A′ and Chain B/B′). Detailed structures of amino acids involved in binding (Glu^A17 and Tyr^A14) are also shown.

Figure 7.

Free energy profiles of phenolic ligands entering the surface binding site III.

Figure 8.

Serotonin binding sites in insulin InsSerT₃R₃ hexamer: top view (top), side view (bottom) of the hexamer (A-chains in pink, B-chains in white, Zn²⁺ ions as blue spheres). Sites I and III are indicated; some N and C termini of A/B-chains (T-state: T, R-state: R) are shown. Serotonin in atom-colored coded, with C-atoms in yellow.

Figure 9.

Serotonin binding sites: top, site I; bottom, site III, in insulin InsSerT₃R₃ complex (insulin color-coded as in Fig. 7, with side-chain C-atoms in green, water as a red sphere). Dashed lines, HB; *R and *T, correspond to symmetry-related dimer and T/R state of the monomer, respectively. Strong His^B5-Nϵ2·π-pyrrole-center contact is also shown.

Figure 10.

Serotonin and arginine binding sites in insulin InsSerArgT₃R₃ hexamer: top view (top) and side view (bottom) of the hexamer (labeling and color-coding as in Fig. 7, with arginine C-atoms in light green).

Figure 11.

Arginine binding sites in the insulin InsSerArgT₃R₃ hexamer. Labeling and color-coding is as in Fig. 8, with Cl⁻ ion as yellow.

Figure 12.

Titration of insulin hexamers and 4H3N with phenol (A) or serotonin (B). Titration of insulin hexamers and 4H3N preincubated with 5 mm arginine with phenol (C) or serotonin (D). Titration of insulin hexamers and 4H3N preincubated with 5 mm dopamine with phenol (E) or serotonin (F). All measured binding curves are shown.