Multiscale Simulations and Profiling of Human Thymidine Phosphorylase Mutations: Insights into Structural, Dynamics, and Functional Impacts in Mitochondrial Neurogastrointestinal Encephalopathy

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a rare metabolic disorder caused by missense mutations in the <i>TYMP</i> gene, leading to the loss of human thymidine phosphorylase (HTP) activity and subsequent mitochondrial dysfunction. Despite its well-characterized biochemical basis, the molecular mechanisms by which MNGIE-associated mutations alter HTP's structural stability, dynamics, and substrate (thymidine) binding remain unclear. In this study, we employ a multiscale computational approach, integrating AlphaFold2-based structural modeling, all-atom and coarse-grained molecular dynamics (MD) simulations, protein-ligand (HTP-thymidine) docking, HTP-thymidine complex simulations, binding free-energy landscape analysis, and systematic mutational profiling to investigate the impact of key MNGIE-associated mutations (R44Q, G145R, G153S, K222S, and E289A) on HTP function. Analyses of our long-duration multiscale simulations (comprising 9 μs coarse-grained, 1.2 μs all-atom apo HTP, and 1.2 μs HTP-thymidine complex MD simulations) and physicochemical properties reveal that while wild-type HTP maintains structural integrity and strong thymidine-binding affinity, MNGIE-associated mutations induce substantial destabilization, increased flexibility, and reduced enzymatic efficiency. Free-energy landscape analysis highlights a shift toward less stable conformational states in mutant HTPs, further supporting their functional impairment. Additionally, the G145R mutation introduces steric hindrance at the active site, preventing thymidine binding and causing off-site interactions. These findings not only provide fundamental insights into the physicochemical and dynamic alterations underlying HTP dysfunction in MNGIE but also establish a computational framework for guiding future experimental studies and the rational design of therapeutic interventions aimed at restoring HTP function.