Structural Determination of the Interaction of H2S and Insulin

Hydrogen sulfide (H2S) has emerged as an important biological signaling molecule. Its interaction with insulin impacts on glucose and lipid metabolisms. However, the molecular mechanisms underlying the cellular effects of H2S have been unsettled. To obtain direct evidence for H2S-induced post-translational modification of insulin molecule, we structurally characterized insulin following incubation with NaHS, an H2S salt, using X-ray crystallography. Insulin crystals were grown using the hanging drop vapor diffusion method and optimized in MES buffer with PEG MME550 and zinc sulfate. X-ray diffraction data were collected at the Canadian Light Source to resolutions between 2.1–2.2 Å. Six datasets were processed, structures solved and refined (representative structure PDB 9MRA; Rwork/Rfree 0.18/0.23). Structural analysis showed electron density corresponding to a sulfur atom near the amide group of Gln4 in chain B, aligning with prior LC-MS predictions. However, N–S distances ranged from 2.35–3.4 Å across the structures, suggesting the possibility of a transient covalent interaction disrupted by radiation damage during data collection. In addition, one structure exhibited a sulfur atom near Glu residues, suggesting secondary or alternative binding sites. All refined structures were predicted to form hexameric assemblies based on PISA analysis. These results provide structural support to H2S-induced post-translational modification of the insulin molecule.