Water-Mediated Inter-Loop Hydrogen Bonds Stabilize Catalytically Competent Loop 6 Conformations in Triosephosphate Isomerase via Molecular Dynamics

Abstract

Triosephosphate isomerase (TIM) is a model (α/β)₈-barrel enzyme that achieves near diffusion-limited catalytic rates through highly coordinated loop motions. In particular, loop 6 (L6) regulates access to the active site by adopting open or closed conformations. Here, we used molecular dynamics simulations to investigate L6 dynamics, and the structural factors required to stabilize its catalytically competent closed state. Our results show that L6 remains predominantly open in both apo and holo forms under unrestrained conditions, suggesting that substrate binding alone is insufficient to maintain closure. To explore this further, we applied two targeted restraint strategies based on water-mediated inter-loop hydrogen bonds, between Ser211 (L7) and Ala169 (L6), that stabilize L6 closed conformation. These restraints effectively shifted the conformational ensemble toward closed and partially closed loop states, enhancing the stability of key catalytic features such as the hydrophobic clamp (ILE170–LEU230) and the catalytic distance (GLU165 – G3P). Additionally, non-productive interactions between Ser211 and the phosphate group of G3P were minimized. This study highlights the value of combining classical MD with restraint-guided sampling to capture functionally relevant loop conformations and provides a generalizable framework for investigating the conformational dynamics of TIM and related enzymes.