Salt Bridge Builder: Using Residue Distances to Predict Salt Bridge Formation

Salt bridges contribute disproportionately to protein folding stability and protein-protein interaction energetics, yet systematic tools for engineering novel salt bridges remain limited. There are several approaches that can quantify the energetics of removing salt bridges between proteins, but no existing tools are available for adding salt bridges at protein interfaces. Here, we introduce Salt Bridge Builder (SBB), a software package that identifies candidate mutation sites for adding interprotein salt bridges using residue distance heuristics derived from large-scale structural data. Using the SKEMPI v2 database, we demonstrate that charged-to-uncharged mutations that disrupt interprotein salt bridges result in binding free energy penalties significantly larger than those of comparable mutations that do not, underscoring the stabilizing role of salt bridges at protein interfaces. We benchmark six residue distance metrics for their ability to predict salt bridge formation and show that the side-chain centroid distance (SCCD) provides the optimal balance between the predictive performance and computational efficiency. Based on these findings, we formulate an efficient algorithm that identifies putative salt bridge-forming mutations while avoiding disruption of existing electrostatic interactions. We apply SBB to the kinesin superfamily and identify kinesin-5 as uniquely enriched in potential salt-bridge-building sites at the microtubule interface. Molecular dynamics simulations of engineered kinesin-5 mutants reveal that only a subset of predicted salt bridges exhibits high occupancy, highlighting the role of local microenvironments in stabilizing engineered electrostatic interactions. Principal component analysis of the residue microenvironment distinguishes high-occupancy salt bridges, suggesting a path toward a priori stability prediction. Long-range electrostatic force calculations further show that selected mutations modulate kinesin-5-microtubule attraction. Together, this work establishes residue-distance-based salt bridge engineering as a viable protein-protein engineering strategy and provides a foundation for future extensions of SBB that incorporate microenvironment-aware stability prediction.