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

Abstract

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. Based on 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.

Distinct antagonist-bound inactive states underlie the divergence in the structures of the dopamine D2 and D3 receptors

“…In marked contrast to the obvious trend toward unwinding of EL2 in all our simulated D2R complexes, in our recent simulations of MhsT, a transporter protein with a region found by crystallography to alternate between helical and unwound conformations 29 , we failed to observe any spontaneous unwinding over a similar simulation timescale (with the longest simulations being ~5-6 µs) when the region was started from the helical conformation 30,31 . This suggests that the C-terminal helical conformation of EL2 represents a higher energy state than the extended conformation, which allows for observation of the transitions in a simulation timescale not usually adequate to sample folding/unfolding events 32 .…”

Monoamine transporters: structure, intrinsic dynamics and allosteric regulation

“…The allosteric site, proposed for SERT almost three decades ago 53 , has been confirmed in subsequent studies of SERT 5,54 and the structural homologs LeuT [55][56][57][58][59] and the NSS MhsT 60 . This site (S2) is more than 10-12 Å away from S1, at the periphery of the EC vestibule, between the EC loops EL4a-EL4b, EL6 and TM1b, TM6a and TM11 6 (Fig.…”

Dynamic extracellular vestibule of human SERT: unveiling druggable potential with novel high-affinity allosteric inhibitors

“…The insights on the allosteric ligand binding at the less conserved S2 site of NSSs are not only essential for our full understanding of the transport mechanism (7,35,36), but may also allow the development of novel therapeutics with the potential of less side effects (15,21). Indeed, the unique therapeutic profile of S-citalopram (S-CIT), including its relatively better patient acceptability (37), may be partially attributed to the additional allosteric binding of S-CIT at the S2 site (38).…”