Conserved water molecules as structural ligands modulating pathogenic variation in human protein binding sites

Abstract Conserved water molecules (CWMs) are tightly bound solvent molecules that occupy well-defined and recurrent positions in protein structures. Although they are known to influence protein stability, function, and ligand binding, their contribution to human genetic disease has remained largely unexplored. Here, we demonstrate that CWMs substantially contribute to the pathogenicity of single nucleotide polymorphisms (SNPs). By systematically mapping SNPs onto ligand-binding and conserved water sites across human protein structures in the Protein Data Bank, we find that pathogenic variants are strongly enriched at CWM positions. Enrichment is particularly pronounced at CWM sites within ligand-binding regions, exceeding that observed for ligand-binding sites as a whole. To establish a mechanistic link, we performed molecular dynamics simulations on human lysosomal acid glucosylceramidase (GCase), encoded by GBA1 and associated with Gaucher disease and Parkinson’s disease risk. Removal of a single conserved water molecule in the wild-type protein recapitulates key structural features of the pathogenic L444P variant, whereas stabilization of this water in the mutant restores native-like behavior. These findings demonstrate that disruption of a conserved water molecule can induce long-range structural changes consistent with disease-associated mutations. Together, our results identify conserved water molecules as functional structural elements whose disruption represents a recurrent mechanism of protein dysfunction and provide direct mechanistic evidence for their pathogenic role in Gaucher disease. Significance Statement Are conserved water molecules hidden determinants of human genetic disease? By systematically mapping single nucleotide polymorphisms (SNPs) onto protein structures, we show that pathogenic variants are strongly enriched at conserved water positions, particularly within ligand-binding sites. This enrichment exceeds that observed for most previously studied ligand types, including small molecules, nucleic acids, and proteins. Molecular dynamics simulations further reveal that disruption of a single conserved water molecule can induce long-range structural effects consistent with disease-associated variants in lysosomal acid glucosylceramidase, linking these findings to Gaucher disease and Parkinson’s disease risk. Together, our results establish conserved water molecules as previously underappreciated structural determinants of human disease and highlight their relevance for understanding genetic variation and guiding drug discovery. Competing Interest Statement The authors have declared no competing interest.