Data for 'Temporal Hydrogen-Bond Network Analysis Reveals Substrate-Directed Connectivity in Dihydrofolate Reductase'

Corresponding Author: Tandac Furkan Guclu ([e-mail]) 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 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.通讯作者：Tandac Furkan Guclu（[e-mail]） 氢键网络（hydrogen-bond networks）是蛋白质功能的核心，但绝大多数网络分析均依赖静态表征，忽略了相互作用随时间的演化过程。本研究提出一种整合瞬时图分析与时序图分析的框架，针对从分子动力学（molecular dynamics）轨迹中提取的氢键网络，量化配体导向的氢键连通性。本研究将该框架应用于大肠杆菌二氢叶酸还原酶（E. coli dihydrofolate reductase, DHFR）及其L28R突变体，计算所有残基至底物二氢叶酸（dihydrofolate, DHF）的最短氢键路径。瞬时分析结果显示，二氢叶酸导向的连通性通过一组稀疏的优选通路构建，野生型酶中的关键枢纽残基为D27与T113。时序分析则凸显出优先支持时序化二氢叶酸导向连通性的残基。与L28R突变体的对比表明，该突变保留了主要的底物接触结构与整体通信骨架，但重新分布了通路的使用模式、持续时长及时序收敛性。网络衍生的热点区域与独立的共演化信号存在部分重叠，其中以K109–I115区域的重叠度最高；而与隐蔽位点（cryptic-site）预测结果的重叠则相对有限。该结果表明，氢键网络能够捕捉到二氢叶酸还原酶中受演化支持的通信区域，而这类区域无法通过静态结构分析方法完全挖掘。本框架可广泛应用于配体结合蛋白，并提供了一条直接从时序化模拟数据中识别持续、延迟及突变敏感型信号通路的路径。