Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue

Zhao, Yongfang; Terry, Daniel S.; Shi, Lei; Quick, Matthias; Weinstein, Harel; Blanchard, Scott C.; Javitch, Jonathan A.

doi:10.1038/nature09971

Cited by 277 publications

(366 citation statements)

References 33 publications

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“…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).…”

supporting

confidence: 78%

“…It has been proposed that the conformational changes required for the alternating-access mechanism involve relative movements of the bundle with respect to elements of the scaffold by the rockingbundle mechanism (11,(15)(16)(17). Other gating-like mechanisms involving local conformational changes (i.e., at the level of individual TM helices) also have been put forward (18,19), and recent studies suggest mechanisms that combine elements of both types (7,20).…”

mentioning

confidence: 99%

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

Khafizov¹,

Pérez²,

Koshy³

et al. 2012

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

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Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K m and K d for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22 Na + binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.secondary transport | symmetry | membrane protein | alkali metal ion | osmoregulation

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supporting

confidence: 78%

mentioning

confidence: 99%

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

Khafizov¹,

Pérez²,

Koshy³

et al. 2012

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

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“…Taken together, our findings provide more evidence against the supposition that there is a second or S2 high-affinity substrate binding site in LeuT [18][19][20][21][22]. The results of the Javitch group related to the S2 site, the properties of the F253A mutant and the role of the putative S2 site in the mechanism of LeuT and NSSs are, at present, without satisfactory explanation.…”

Section: Discussionmentioning

confidence: 49%

“…By contrast, molecular dynamics, substrate binding and singlemolecule experiments have been interpreted in terms of a model in which there is a second, or S2, high-affinity substrate binding site [18][19][20][21][22]. In an effort to disrupt substrate binding to the S1 site, Javitch and colleagues [18,21] studied the F253A mutant, a residue that lines a portion of the S1 binding site [6,14]. On the basis of their experiments on the F253A mutant, the authors make several assertions.…”

Section: Introductionmentioning

confidence: 99%

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

Wang

Gouaux

2012

EMBO Reports

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LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.

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“…In LeuT, a phenylalanine in the corresponding EH2 segment (EL4) was proposed to contribute to the formation of a periplasmic second substrate-binding (S2) site (44). The S2 site is assumed as being transiently occupied during translocation as the substrate moves towards the primary substrate-binding (S1) site (45). Formation of a S2 site in BetP has not been structurally corroborated although a periplasmic S2 site was reported for the homologous antiporter CaiT (46), a nonregulated member of the BCCT-family (6).…”

Section: Superimposition Of F-d Curves Recorded At Buffer Conditionmentioning

confidence: 99%

Locating an extracellular K⁺-dependent interaction site that modulates betaine-binding of the Na⁺-coupled betaine symporter BetP

Pérez

Waclawska

et al. 2011

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

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BetP, a trimeric Na þ -coupled betaine symporter, senses hyperosmotic stress via its cytoplasmic C-terminal domain and regulates transport activity in dependence of the cytoplasmic K þ -concentration. This transport regulation of BetP depends on a sophisticated interaction network. Using single-molecule force spectroscopy we structurally localize and quantify these interactions changing on K þ -dependent transport activation and substrate-binding. K þ significantly strengthened all interactions, modulated lifetimes of functionally important structural regions, and increased the mechanical rigidity of the symporter. Substrate-binding could modulate, but not establish most of these K þ -dependent interactions. A pronounced effect triggered by K þ was observed at the periplasmic helical loop EH2. Tryptophan quenching experiments revealed that elevated K þ -concentrations akin to those BetP encounters during hyperosmotic stress trigger the formation of a periplasmic second betaine-binding (S2) site, which was found to be at a similar position reported previously for the BetP homologue CaiT. In BetP, the presence of the S2 site strengthened the interaction between EH2, transmembrane α-helix 12 and the K þ -sensing C-terminal domain resulting in a K þ -dependent cooperative betaine-binding.atomic force microscopy | energy landscape | membrane transporter | osmoregulation | secondary substrate-binding site

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Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue

Cited by 277 publications

References 33 publications

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

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

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

Locating an extracellular K⁺-dependent interaction site that modulates betaine-binding of the Na⁺-coupled betaine symporter BetP

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Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue

Cited by 277 publications

References 33 publications

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

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

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+-coupled betaine symporter BetP

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Locating an extracellular K⁺-dependent interaction site that modulates betaine-binding of the Na⁺-coupled betaine symporter BetP