Temporal Hydrogen‐Bond Network Analysis Reveals Substrate‐Directed Connectivity in Dihydrofolate Reductase

ABSTRACT Hydrogen‐bond networks are central to protein function, but most network analyses rely on static representations that neglect how interactions evolve in time. Here, we introduce a framework that combines instantaneous and temporal graph analysis of hydrogen‐bond networks derived from molecular dynamics (MD) trajectories to quantify ligand‐directed hydrogen‐bond connectivity. We apply the method to E. coli dihydrofolate reductase (DHFR) and its L28R mutant, computing shortest hydrogen‐bond paths from all residues to the substrate dihydrofolate (DHF). The instantaneous analysis reveals that DHF‐directed connectivity is organized through a sparse set of preferred routes, with D27 and T113 acting as prominent hubs in the wild‐type enzyme. Temporal analysis highlights residues that preferentially support time‐ordered DHF‐directed connectivity. Comparison with L28R shows that the mutation preserves the main substrate‐contacting architecture and the overall communication scaffold but redistributes pathway usage, persistence, and temporal convergence. The network‐derived hotspots partially overlap with independent coevolution signals, most strongly in the K109–I115 region, while overlap with cryptic‐site predictors is more limited. This pattern indicates that the hydrogen‐bond network captures evolutionarily supported communication regions in DHFR that are not fully recovered by static structural approaches. The framework is broadly applicable to ligand‐binding proteins and provides a route to identify persistent, delayed, and mutation‐sensitive signaling pathways directly from time‐ordered simulation data.