The Systematic Study of Spatially Conserved Salt Bridges in Protein.

Salt-bridge conservation has traditionally been evaluated at the primary sequence level, leaving the persistence of three-dimensional interaction sites across homologous families largely unexplored. In this study, we developed a systematic family-level structural framework to redefine salt-bridge conservation based on spatial interaction sites rather than residue identity by mapping salt bridges onto unified SCOP2-aligned domain coordinates. We classified these interactions into three categories: classically conserved (CLA), nonclassically conserved (NOCLA; i.e., charge compensation), and nonconserved. This spatial definition enabled the identification of charge-swapped interactions that are invisible to standard sequence alignment. Through a comprehensive analysis of 5,679 protein families, we demonstrated that charge compensation is a recurrent and evolutionarily preserved mode of spatial salt-bridge conservation across homologous families. NOCLA was not uniformly distributed but instead showed marked structural-context dependence, being preferentially enriched in alpha and beta proteins (a/b) and concentrated in a limited subset of folds, particularly protein kinase-like, PLP-dependent transferase-like, TIM beta/alpha-barrel, and globin-like folds. To assess functional relevance, we integrated variant-effect predictors, including AlphaMissense and ESM-1v. Our results revealed that spatially conserved salt bridges exhibited significantly higher mutational sensitivity and functional constraint than nonconserved sites (CLA > NOCLA > nonconserved). Notably, highly sensitive NOCLA positions were also the most structurally concentrated, arising predominantly from a restricted set of folds, especially protein kinase-like folds, in contrast to the broader distribution of CLA and the highly dispersed pattern of nonconserved sites. Furthermore, molecular dynamics (MD) simulations coupled with MDPath-based mutual-information network analysis demonstrated that disruption of a representative NOCLA site significantly reorganizes long-range communication pathways within conserved catalytic regions of kinase domains. These findings suggest that spatially conserved salt bridges serve not only as local electrostatic stabilizers but also as critical dynamic coupling nodes within protein structures. Together, this study provides a three-dimensional family-level paradigm for analyzing electrostatic interactions in protein evolution and offers new mechanistic insights for interpreting variant effects and guiding structure-based drug design.