X-ray Crystallographic and Hydrogen Deuterium Exchange Studies Confirm Alternate Kinetic Models for Homolog Insulin Monomers

Abstract

Despite the crucial role of various insulin analogs in achieving satisfactory glycemic control, a comprehensive understanding of their dynamic mechanisms still holds the potential to further optimize ultra-fast-acting insulin analogs, thus significantly improving the well-being of individuals with Type 1 Diabetes. Here, we present new insights derived from our 2.5 Å resolution X-ray crystal structure of modified insulin analog at ambient temperature. Furthermore, we obtained a distinctive side-chain conformation of the Asn3 residue on the B chain (AsnB3) by operating a comparative analysis with a previously available cryogenic rapid-acting insulin structure (PDB_ID: 4GBN). Additionally, we employed hydrogen-deuterium exchange mass spectrometry to decipher the molecular dynamics of these insulin analogs. A comparative analysis of H/D dynamics demonstrated that the modified insulin exchanges deuterium atoms faster and more extensively than the intact insulin aspart. The hybrid structural approach combined with computational analysis employed in this study provides novel insight into the structural dynamics of newly modified insulin and insulin aspart monomeric entities. It allows further molecular understanding of intermolecular interrelations driving dissociation kinetics and, thus, a fast action mechanism.