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, T3Rf3, 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.

Can Arginine inhibit Insulin Aggregation? A Combined Protein Crystallography, Capillary Electrophoresis, and Molecular Simulation Study

“…On top of this, the carboxylate group of the arginine is coordinating the zinc ion cofactor of the hexamer. Therefore this arginine binding mode corresponds to the starting crystallographic structure, 21 since the structure remained stable, and did not evolve significantly during the course of the simulation. Figure 7.…”

Can Arginine inhibit Insulin Aggregation? A Combined Protein Crystallography, Capillary Electrophoresis, and Molecular Simulation Study

“…To assess the stability of the experimentally observed binding site of arginine, we also performed simulations at minimal ionic strength conditions of the crystallographic complex obtained in this work, which consists of an insulin dimer with one arginine in each of its monomers (System I). Since a crystallographic complex of the insulin hexamer exhibiting insulin-bound arginine molecules was previously obtained in the presence of arginine and serotonin, 21 we also explored the stability of the arginine binding site within the insulin hexamer. To this end, we performed simulations at minimal ionic strength conditions (i.e., only compensation of the protein charge) starting from the crystallographic structure (PDB 5MT9), after removal of the crystallographic serotonin molecules (system V).…”

“…Since MD simulations as well as capillary electrophoresis and previous crystallographic experiments 21 indicated that insulin hexamerization appears to be possible in the presence of arginine at least at low ionic strength, we also probed the arginine binding to the insulin hexamer using MD simulations. Firstly, we assessed the stability of the crystallographic binding site for arginine in a T3R f 3 hexamer:serotonin:arginine complex.…”

“…There, arginine penetrated the insulin T3R f 3 hexamer filling the voids around Zn 2+ and HisB10. 21 Arginine is a biogenic molecule that may be abundant in the granules upon processing of proinsulin at its two arginine-rich sites. 22 Its presence in insulin solution is also of a clinical relevance as small arginine-rich protamines are employed in some injection formulations of this hormone.…”

“…corresponds to the starting crystallographic structure,21 since the structure remained stable, and did not evolve significantly during the course of the simulation.…”