Molecular Mechanism of pH-Dependent Activation in Human Equilibrative Nucleoside Transporter 3

Human equilibrative nucleoside transporter 3 (hENT3) is a lysosomal transporter whose activity is strictly dependent on an acidic lumenal environment. Mutations in hENT3 lead to H syndrome and other metabolic disorders, yet the structural basis for its pH-sensitive gating remains poorly defined. Using multi-replica conventional MD simulations, we provide atomistic insight into the pH-dependent activation mechanism of hENT3 in the outward-facing state. Protonated Asp219 triggers a local hydrogen-bond reorganization that recruits conserved Ser229 and the TM6 helix-capping residue Asp225 and stabilizes TM5-an intrinsically flexible helix within the ENT family. Stabilization of TM5 in turn reinforces the neighboring helices including TM1, TM6 and TM8, reduces thin-gate fluctuations, and more frequently preserves the adenosine binding pose required for translocation. On the cytosolic side, Glu447 protonation modulates the interaction between the H-syndrome-associated Arg134 and loop 2. With an alignment-independent distance correlation analysis, we identified distinct state-dependent coupling patterns under acidic and neutral pH conditions. In particular, acidic ensembles that retained a transport-competent binding pose showed the strongest agreement with prior mutagenesis data, as site-centric distance correlation scores are consistent with experimentally observed loss of adenosine transport.