Nanoscopic Origin of Enhanced Tyrosine-Mediated Water Ordering over Canonical Threonine Motifs in a Bacterial Ice-Nucleating Protein

Bacterial ice-nucleating proteins (INPs) are known as the most efficient biological heterogeneous ice nucleators. Ice-nucleation ability is conventionally attributed to the threonine-based conserved motifs of {beta} -helical INPs, whereas the role of other conserved motifs remains overlooked. To identify the motif with the highest hydration-ordering ability, a 16-loop model INP and its two mutants are simulated using all-atom molecular dynamics at 260 K, just below the ice nucleation temperature, using the CHARMM36m force field and TIP4P/Ice water model. Among four potential ice-nucleating sites present in four exposed surfaces of the {beta} -helical INP, the glycine-tyrosine-glycine (GYG) motif-associated interface water (IW) exhibits the probability distribution of the Steinhardt bond-orientational and tetrahedral order parameters closest to that of the Ih. The GYG arrange themselves into a ladder-like array of tyrosine hydroxyls, compatible with the Ih-like lattice geometry, suggesting that this motif provides the IW a favorable template for ice-like water organization. The Y-to-phenylalanine mutation produces the largest disruption in IW order compared to the threonine mutations. Although the difference in entropy of threonine-associated IW and Ih is the least among all sites, ice-like hydration ordering near GYG may exceed prior assumptions for a threonine-based ice-nucleating surface. The ordering is collectively determined by lattice compatibility, entropy reduction, hydrophobicity, interfacial geometry, and sequence conservation. The study unfolds a molecular framework of biological ice nucleation and provides a foundation for the rational engineering of more efficient artificial ice-nucleators for applications in climate-related technologies.