The role of transmembrane segment 5 (TM5) in Na2 release and the conformational transition of neurotransmitter:sodium symporters toward the inward-open state

Abstract

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by the reuptake of released neurotransmitters. This active accumulation of substrate against its concentration gradient is driven by the transmembrane Na gradient and requires that the transporter traverses several conformational states. LeuT, a prokaryotic NSS homolog, has been crystallized in outward-open, outward-occluded, and inward-open states. Two crystal structures of another prokaryotic NSS homolog, the multihydrophobic amino acid transporter (MhsT) from , have been resolved in novel inward-occluded states, with the extracellular vestibule closed and the intracellular portion of transmembrane segment 5 (TM5i) in either an unwound or a helical conformation. We have investigated the potential involvement of TM5i in binding and unbinding of Na2, the Na bound in the Na2 site, by carrying out comparative molecular dynamics simulations of the models derived from the two MhsT structures. We find that the helical TM5i conformation is associated with a higher propensity for Na2 release, which leads to the repositioning of the N terminus and transition to an inward-open state. By using comparative interaction network analysis, we also identify allosteric pathways connecting TM5i and the Na2 binding site to the extracellular and intracellular regions. Based on our combined computational and mutagenesis studies of MhsT and LeuT, we propose that TM5i plays a key role in Na2 binding and release associated with the conformational transition toward the inward-open state, a role that is likely to be shared across the NSS family.

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 .…”

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

“…To quantify such changes, we calculated structural differences at both subsegment and residue levels between the most dominant green MS and the S2: apo condition (the data of which were extracted from ref 17 ) using pairwise interaction analyzer for MhsT (PIA-MhsT, see Methods). At the subsegment level, the results of our analysis indicate that the most prominent changes are the rearrangement of EL5 and TM10e on the extracellular side (Figure 2A).…”

“…(A) The pairwise distances among TM subsegments in the green MS are compared to those in the S2: apo condition. 17 The analysis highlights the conformational changes of TM10e and the neighboring EL5 in the green MS. (B) The pairwise Cα atom distances of the S2:Trp-interacting residues identified in the green MS (Table 1) are compared to those in the S2: apo condition. The results indicate significant changes of residues Phe366 (EL5) and Phe381 (TM10e) (see text).…”

“…The recent crystal structures of MhsT (PDB IDs: 4US3 and 4US4), 16 another bacterial NSS, were solved in an inward-occluded conformation, 17 with a substrate (L-Trp)-occupied S1 site and a collapsed EV. Saturation binding studies in n-dodecyl-β-D-maltopyranoside (DDM), the detergent used for the crystallization of MhsT, supported a molar binding stoichiometry of 1.…”

Cholesterol binding to a conserved site modulates the conformation, pharmacology, and transport kinetics of the human serotonin transporter

“…The location of CHOL1 at the interface between the scaffold (TM5) and the conformationally flexible bundle (TM1a and TM7) (19) (see Figure 1) makes it an interesting candidate as a specific cholesterol binding site that modulates the conformational dynamics of the transporters. TM1a is of particular interest in the conformational role of cholesterol because this region has been shown to undergo large conformational changes in LeuT and MhsT during the transition from outward-facing to inwardfacing conformation (20)(21)(22)(23)(24). Similarly, TM5 has been shown to line the intracellular permeation pathway in SERT and be more accessible in the inward-facing conformation (25,26) and may also respond to cholesterol binding at CHOL1 with a conformational change.…”