Single-molecule dynamics of gating in a neurotransmitter transporter homologue

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

doi:10.1038/nature09057

Cited by 248 publications

(403 citation statements)

References 52 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%

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 recent years, it has, however, become apparent that transporters show conformational dynamics and that those have to be understood and characterized (Locher, 2016). For this, single‐molecule Förster resonance energy transfer (smFRET; Lerner et al , 2018) has been used to monitor these conformational dynamics of transporters, e.g., the P‐type Ca 2+ ‐ATPase (LMCA1) from Listeria monocytogenes (Dyla et al , 2017), secondary transporters such as the aspartate/Na + symporter from Pyrococcus horikoshii (Akyuz et al , 2013), and the leucine/Na + symporter LeuT from Aquifex aeolicus (Zhao et al , 2010). While there are also smFRET studies of ABC importers regarding the conformational dynamics of the substrate binding domains (SBDs; Gouridis et al , 2015) or interactions of SBD‐TMD (Goudsmits et al , 2017a; Yang et al , 2018), there are no studies of conformational dynamics and crosstalk between TMDs and NBDs in ABC exporters in native‐like lipid environment (Verhalen et al , 2012).…”

Section: Introductionmentioning

confidence: 99%

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

et al. 2018

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ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward‐open conformation) and hydrolysis (inward‐open) for unidirectional transport driven by ATP. Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single‐molecule Förster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native‐like lipid environment and directly monitor conformational dynamics in both the transmembrane‐ (TMD) and nucleotide‐binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.

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Single-molecule dynamics of gating in a neurotransmitter transporter homologue

Cited by 248 publications

References 52 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

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

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