Conformational Rearrangements to the Intracellular Open States of the LeuT and ApcT Transporters Are Modulated by Common Mechanisms

Abstract

Recent crystallographic studies revealed that five transporter families without much sequence similarities among them have similar structure folds to LeuT, a bacterial neurotransmitter:sodium symporter homolog. The LeuT fold is characterized by an internal twofold structural pseudosymmetry. The transport cycle of some members of each of these families is dependent on a sodium gradient across the membrane, whereas in some others the role of sodium is mimicked by proton. We report on the identification of common structure-dynamics elements of the transporters with LeuT fold, which are recognizable in the conformational transitions related to function. The findings from comparative computational modeling and simulation studies of LeuT, and ApcT from the amino acid-polyamine-organocation transporter family define the intramolecular mechanisms by which Na+ binding couples to the transport process, and single out the lead/active role of TM1a in the transition to inward-open conformation. These mechanistic insights are derived in the context of collaborative investigations of LeuT dynamics with both single-molecule fluorescence and simulations that have produced excellent agreement of the dynamic details, and are found to be generalizable across the transporter families and to transcend sequence and motif similarities.

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

“…2) (12,21). These two sites are occupied in two states of LeuT open to the outside (12,20), consistent with the notion that sodium binds preferentially to, and presumably stabilizes, outward-facing conformations of the FIRL-fold, as shown by spectroscopic measurements of the effect of sodium on LeuT (18,19,22) and by molecular dynamics (MD) simulations of LeuT in the absence of the ion at Na2, during which the cytoplasmic pathway begins to open (23,24).…”

“…1 and 2) (9). Protonation and deprotonation of K158 were proposed to play the same role as binding of Na2 in LeuT (9), indicating a common mechanistic principle, which was supported by subsequent MD simulations (23). Similarly, in the carnitine:γ-butyrobetaine antiporter CaiT, which also is neither sodium-nor proton-dependent, an arginine residue (R262) located at a position equivalent to Na2 bridges T100 in A1 and T421 in B3 (Figs.…”

Structure-based ligand discovery for the Large-neutral Amino Acid Transporter 1, LAT-1

“…Structures of the arginine:agmatine antiporter AdiC from Escherichia coli (15,17,18) and Salmonella enterica (20) in different conformations reveal an internal twofold pseudosymmetry, similar to the structures of the sodium-and chloride-dependent leucine transporter, LeuT (19,21). These data, combined with structures of additional related transporters (22) and molecular dynamics (MD) simulations (23), suggest a common transport mechanism among the LAT-1 homologs and LeuT, in which the role of sodium in LeuT is proposed to be mimicked by a proton in some APC transporters (23). Thus, LAT-1 probably also transports ligands across the cell membrane via the alternating access transport mechanism (22,24,25).…”

Steric Hindrance Mutagenesis in the Conserved Extracellular Vestibule Impedes Allosteric Binding of Antidepressants to the Serotonin Transporter

“…Its practical advantage is the increased sampling of binding modes so as to overcome the difficulties presented by the large molecular sizes of the inhibitors of SERT and their relatively low affinities for the S2 site. Moreover, the iterative relaxation and sampling through MD reduce the time required for convergence and the uncertainty associated with the use of a homology model (36) for SERT.…”

“…The iterative approach is briefly illustrated for the SERT configuration with (S)-CIT in the S1 site and CMI in the S2 site: (i) an (S)-CIT molecule was first docked into the S1 site of the SERT model (36); (ii) the top ranked ligand-transporter complex was reimmersed in an all-atom representation of a bilayerwater environment and was subjected to 48-ns MD simulations; (iii) a CMI molecule was subsequently docked in the S2 site, based on an equilibrated MD snapshot from stage (ii); and (iv) an MD simulation in explicit solvent was carried out following the protocol described in (ii). For this particular case, we observed a significant rearrangement of the interactions between the transporter and the CMI ligand that moved from its initial docking pose (ϳ2-Å translation).…”