Exploring Ligand-Binding Kinetics in the S2 Site of MhsT by Atomistic Simulations and Markov Models

Abstract

Neurotransmitter:Sodium Symporters (NSS) terminate neurotransmission through sodium-driven reuptake of cognate neurotransmitters, and traverse between outward-open and inward-open states. NSS include serotonin and dopamine transporters, which are targets for antidepressants and abused psychostimulants. Crystallographically, whereas both substrates and inhibitors have been found to bind the central binding site (denoted S1) of the NSS proteins, it has been shown that inhibitors for LeuT and SERT can also bind to a binding cavity in the extracellular vestibule (denoted S2). Based on computational and experimental studies in LeuT, it has been found that substrates can bind to S2 as well, and such binding triggers the intracellular release the substrate and Na+ from S1. However, whether such a role of S2 substrate is common for other NSS remains unclear.The newly available crystal structure of MhsT, a bacterial NSS homolog, which was solved in an inward-occluded state, provides a more suitable starting point to study the substrate induced conformational changes, including the S2 substrate induced transition from inward-occluded to inward-open state transition. Here, we use extensive molecular dynamics simulations combined with hidden Markov model (HMM) analysis to investigate the feasibility and kinetics of the substrate L-tryptophan binding to S2 of MhsT. Based on HMM analysis, we identified transition binding positions in the extracellular vestibule that form potential substrate binding pathways, and the conformational changes near the S2 site associated with the two most populated bound states. Our findings shed light on the S2 substrate binding kinetics and the induced conformational changes, which may be critical in triggering the subsequent events in the transport mechanism.