Cavity Waters Govern Insulin Association and Release: Inferences from Experimental Data and Molecular Dynamics Simulations

TL;DR

This study combines experimental data and molecular dynamics simulations to reveal that water molecules within the insulin hexamer cavity are crucial for its structural stability and regulation of its dissociation into monomers.

Contribution

It provides new insights into the role of cavity water molecules in stabilizing insulin hexamer structure through hydrogen bonding and electrostatic interactions, supported by experimental and simulation data.

Findings

Cavity water molecules stabilize the hexamer structure.

Removal of cavity waters causes collapse of the hexamer.

Hydrogen bond network is more rigid inside the cavity.

Abstract

While a monomer of the ubiquitous hormone insulin is the biologically active form in the human body, its hexameric assembly acts as an efficient storage unit. However, the role of water molecules in the structure, stability and dynamics of the insulin hexamer is poorly understood. Here we combine experimental data with molecular dynamics simulations to investigate the shape, structure and stability of an insulin hexamer focusing on the role of water molecules. Both X-Ray analysis and computer simulations show that the core of the hexamer cavity is barrel-shaped, holding, on an average, sixteen water molecules. These encapsulated and constrained molecules impart structural stability to the hexamer. Apart from the electrostatic interactions with Zn2+ ions, an intricate hydrogen bond network amongst cavity water and neighboring protein residues stabilizes the hexameric association. These water molecules solvate six glutamate residues inside the cavity decreasing electrostatic repulsions amongst the negatively charged carboxylate groups. They also prevent association between glutamate residues and Zn2+ ions and maintain the integrity of the cavity. Simulations reveal that removal of these waters results in a collapse of the cavity. Subsequent analyses also show that the hydrogen bond network among these water molecules and protein residues that face the inner side of the cavity is more rigid with a slower relaxation as compared to that of the bulk solvent. Dynamics of cavity water reveal certain slow water molecules which form the back bone of the stable hydrogen bond network. The analysis presented here suggests a dominant role of structurally conserved water molecules in maintaining the integrity of the hexameric assembly and potentially modulating the dissociation of this assembly into the functional monomeric form.