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

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.