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

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.