Local Conformational Changes in the <i>Vibrio</i> Na<sup>+</sup>/Galactose Cotransporter

Veenstra, Monique; Lanza, Seren; Hirayama, Bruce A.; Turk, Eric; Wright, Ernest M.

doi:10.1021/bi0357210

Cited by 17 publications

(25 citation statements)

References 25 publications

(39 reference statements)

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“…For hSGLT1, two Na + ions bind to the empty transporter before glucose. The order of binding to the two Na + sites (Na1, Na2) is not known, but the available evidence tends to favor Na + binding first to the Na2 site: First, the Na2 site is conserved in vSGLT and hSGLT2, and these proteins couple only one Na + to sugar transport (42)(43)(44); second, MD studies on LeuT have led to the predictions that Na + binds first to the Na2 site and then to the Na1 site even in the absence of substrate (45); third, Na + has also been proposed to bind first to the Na2 site in BetP (32); and fourth, Na + is proposed to bind first to the Na2 site on the human GABA transporter GAT-1, followed by cooperative binding of the second Na + to the Na1 site (46). In all cases Na + binding stabilizes the transporter in an outward-open conformation to facilitate substrate binding, but it is not yet clear whether substrate binding increases the Na1 affinity for Na + .…”

Section: Resultsmentioning

confidence: 99%

Functional identification and characterization of sodium binding sites in Na symporters

Loo

Jiang

Gorraitz

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na + in binding sites is beyond the resolution of the structures, two Na + binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (K d ) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na + binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two -OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium K d by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na + and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na + binding, and that Na + binding to Na2 promotes binding to Na1 and also sugar binding.I on coupled symporters, or cotransporters, such as hSGLT1 use electrochemical potential gradients to drive solutes into cells. A common finding for these transporters is that external Na + binds before the substrate. Na + binding induces a conformational change of the protein, resulting in the substrate vestibule becoming open to the external membrane surface. After substrate binding, the two ligands are transported across the membrane and are released into the cytoplasm. The atomic structures of several sodium dependent transporters have been solved [leucine transporter (LeuT), vibrio parahaemolyticus sodium glucose (vSGLT), sodium hydantoin (Mhp1), and sodium betaine (BetP)] (1-6). They share a common structural fold with a five-helix inverted repeat, the "LeuT fold". The substrate binding sites are located in the middle of the protein, isolated from the external and membrane surfaces by hydrophobic gates, and putative Na + sites have been identified. Testing these binding sites is a problem due to the fact that phenomenological kinetic constants for ligand transport (K 0.5 , half-saturation values) are interdependent on each other (7). Here we have developed a method to estimate the intrinsic Na + dissociation constants (K d ) for human sodium glucose cotransport (hSGLT1) that may be broadly applicable to other symporters. We then use this to investigate the importance of hSGLT1 residues predicted to be at or near the two Na + binding sites, Na1 and Na2.The method is based on the fact that voltage-dependent membrane proteins exhibit transient charge movements (capacitive transients) i...

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Section: Resultsmentioning

confidence: 99%

Functional identification and characterization of sodium binding sites in Na symporters

Loo

Jiang

Gorraitz

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Sodium is the ion driving substrate translocation in LeuT, vSGLT, Mhp1 and BetP, however the sodium to substrate stoichiometry may differ amongst these sodium dependent transporters: LeuT and BetP have a 2:1 stoichiometry [8,22,23]; and vSGLT and Mhp1 have a 1:1 stoichiometry [7,25] [7]. In the founding structure of LeuT (at 1.65Å), two likely sodium sites (Na1 and Na2) were identified [5].…”

Section: Sodium Binding Site(s)mentioning

confidence: 99%

“…The complete cycle results in the transport of sodium and substrate across the membrane in a fixed stoichiometry (1/1, 2/1). The direction of transport is determined by the ligand concentrations on each side of the membrane and the membrane potential [14,25,27,31-33,43]. The crystal structures of Mhp1 appear to correspond to C2 and C3, LeuT to C3, BetP to C4 and vSGLT to C5.…”

Section: Figurementioning

confidence: 99%

Structure and function of Na+-symporters with inverted repeats

Abramson

Wright

2009

Current Opinion in Structural Biology

182

180

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Summary Symporters are membrane proteins that couple energy stored in electrochemical potential gradients to drive the cotransport of molecules and ions into cells. Traditionally, proteins are classified into gene families based on sequence homology and functional properties, e.g. the sodium glucose (SLC5 or Sodium Solute Symporter Family, SSS or SSF) and GABA (SLC6 or Neurotransmitter Sodium Symporter Family, NSS or SNF) symporter families [1-4]. Recently, it has been established that four Na+-symporter proteins with unrelated sequences have a common structural core containing an inverted repeat of 5 transmembrane (TM) helices [5-8]. Analysis of these four structures reveals that they reside in different conformations along the transport cycle providing atomic insight into the mechanism of sodium solute cotransport.

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“…Although the sequence of binding events from the extracellular side has been partially characterized for vSGLT and hSGLT1 (the homologous human Na + /glucose transporter) experimentally (26–28), the sequence and the mechanism of unbinding of the transported species from the protein into the cytoplasmic side are largely unknown. While the presence of the co-transported Na + ion in the protein could not be unequivocally determined using the experimental data, in the reported PDB file, a Na + ion was modeled in a putative binding site corresponding to that of the homologous protein LeuT based on the structural alignment and mutagenesis results (11).…”

Section: Introductionmentioning

confidence: 99%

A gate-free pathway for substrate release from the inward-facing state of the Na+-galactose transporter

Tajkhorshid

2012

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Employing molecular dynamics (MD) simulations, the pathway and mechanism of substrate unbinding from the inward-facing state of the Na+–coupled galactose transporter, vSGLT, have been investigated. During a 200–ns equilibrium simulation, repeated spontaneous unbinding events of the substrate from its binding site have been observed. In contrast to the previously proposed gating role of a tyrosine residue (Y263), the unbinding mechanism captured in the present equilibrium simulation does not rely on the displacement and/or rotation of this side chain. Rather, the unbinding involves an initial lateral displacement of the substrate out of the binding site which allows the substrate to completely emerge from the region covered by the side chain of Y263 without any noticeable conformational changes of the latter. Starting with the snapshots taken from this equilibrium simulation with the substrate outside the binding site, steered MD (SMD) simulations were then used to probe the translocation of the substrate along the remaining of the release pathway within the protein’s lumen and to characterize the nature of protein-substrate interactions involved in the process. Combining the results of the equilibrium and SMD simulations, we provide a description of the full translocation pathway for the substrate release from the binding site into the cytoplasm. Residues E68, N142, T431, and N267 facilitate the initial substrate’s displacement out of the binding site, while the translocation of the substrate along the remainder of the exit pathway formed between TM6 and TM8 is facilitated by H–bond interactions between the substrate and a series of conserved, polar residues (Y138, N267, R273, S365, S368, N371, S372, and T375). The observed molecular events indicate that no gating is required for the release of the substrate from the crystallographically captured structure of the inward-facing state of SGLT, suggesting that this conformation might represent an open, rather than occluded, state of the transporter.

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Local Conformational Changes in the Vibrio Na⁺/Galactose Cotransporter

Cited by 17 publications

References 25 publications

Functional identification and characterization of sodium binding sites in Na symporters

Functional identification and characterization of sodium binding sites in Na symporters

Structure and function of Na+-symporters with inverted repeats

A gate-free pathway for substrate release from the inward-facing state of the Na+-galactose transporter

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Local Conformational Changes in the Vibrio Na+/Galactose Cotransporter

Cited by 17 publications

References 25 publications

Functional identification and characterization of sodium binding sites in Na symporters

Functional identification and characterization of sodium binding sites in Na symporters

Structure and function of Na+-symporters with inverted repeats

A gate-free pathway for substrate release from the inward-facing state of the Na+-galactose transporter

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Local Conformational Changes in the Vibrio Na⁺/Galactose Cotransporter