Visualize Transient Water-Mediated Hydrogen Bonds Facilitating the Formation of Enzymatic Near-Attack Conformers

Water-mediated hydrogen bonds are vital to various macromolecular activities, particularly in enzymatic reactions. However, the dynamic nature of these bonds poses challenges for their detection. Currently, there is a lack of methods for directly resolving residues within proteins that form hydrogen bonds with water at the atomic level. Herein, we combined supercooling techniques with an NMR method based on spin transverse relaxation perturbation through rational manipulations of dipolar interactions and quantum coherence to characterize transient water-mediated hydrogen bonds in proteins. After thorough validation on different proteins, we applied this method to the catalysis of adenylate kinase (AdK) from Escherichia coli. In conjunction with molecular dynamics simulations, we discovered that the formation of water-mediated hydrogen bonds between Q28 and G14 facilitates the configuration of enzymatic near-attack conformations (NACs), providing experimental support for the theoretical framework. Our findings present a methodology for studying transient weak chemical bonds in macromolecules, thereby bridging the gap between molecular dynamics simulations and experimental validation.