Abstract
The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ~1. In addition, the related and more experimentally tractable SSS member PutP (the Na(+)/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport.
Highlights
The solute-sodium symporter (SSS) vSGLT and the neurotransmitter-sodium symporter (NSS) LeuT have similar structural folds, their crystallographically identified substrate sites diverge in location and composition
When we superimposed LeuT and vSGLT with the core TM regions (TM1–10 of LeuT and TM1Ј–10Ј of vSGLT; Fig. 1), we found that the substrate-binding site of vSGLT, which is formed by residues from TM1Ј, -2Ј, -6Ј, -7Ј, and -10Ј, is situated more extracellularly than the central occluded substrate (S1) site in LeuT, which is enclosed by TM1, -3, -6, and -8 (Fig. 2)
Whereas the S2 site in LeuT is located in the middle of the so-called extracellular vestibule (EV) that is composed of residues shown to participate in binding tricyclic antidepressants [15] and selective serotonin reuptake inhibitors [41] in outward-facing conformations, the substrate-binding site of vSGLT is at the intracellular end of the collapsed EV in the inward-facing conformation
Summary
The solute-sodium symporter (SSS) vSGLT and the neurotransmitter-sodium symporter (NSS) LeuT have similar structural folds, their crystallographically identified substrate sites diverge in location and composition. We hypothesized that members of the SSS family in distinct states may bind substrate in additional binding sites, which may not be readily identified with crystallographic approaches To this end, by using a combined approach encompassing computational modeling and different substrate binding assays, we show that two SSSs, vSGLT and the homologous Naϩ/proline transporter (PutP), can simultaneously bind two substrate molecules under equilibrium conditions in the presence of Naϩ, like LeuT of the neurotransmitter-sodium symporter family [24]. In PutP, an experimentally tractable SSS system for which the assessment of different modes of transport has been established [31], these mutations result in severe reduction of proline translocation
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