Exploring Substrate Binding in the Extracellular Vestibule of MhsT by Atomistic Simulations and Markov Models

Neurotransmitter:sodium symporters (NSS) terminate neurotransmission through Na⁺-driven reuptake of cognate neurotransmitters. Crystallographically, whereas both substrates and inhibitors have been found to bind in the central binding (S1) site of NSS, inhibitors were found to bind to a second binding (S2) site in the extracellular vestibule (EV) of transporters for leucine (LeuT) and serotonin. On the basis of computational and experimental studies, we proposed that substrates bind to the S2 site of LeuT as well and that substrate binding to the S2 site is essential for Na⁺-coupled symport. Recent binding experiments show that substrate (l-Trp) binding in the S2 site of MhsT, another bacterial NSS, is also central to the allosteric transport mechanism. Here, we used extensive molecular dynamics simulations combined with Markov state model analysis to investigate the interaction of l-Trp with the EV of MhsT and identified potential binding poses of l-Trp as well as induced conformational changes in the EV. Our computational findings were validated by experimental mutagenesis studies and shed light on the ligand binding characteristics of the EV of NSS, which may facilitate development of allosteric ligands targeting NSS.