Role of Ser-340 and Thr-341 in Transmembrane Domain IX of the Na+/Proline Transporter PutP of Escherichia coli in Ligand Binding and Transport

Hilger, Daniel; Böhm, Maret; Hackmann, Alexandra; Jung, Heinrich

doi:10.1074/jbc.m706741200

Cited by 26 publications

(39 citation statements)

References 39 publications

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“…2). Binding to this site is affected by mutagenesis of S365 in vSGLT to alanine and of analogous residues in other SSS proteins (29)(30)(31). Consistent with the observations for Mhp1, MD simulations of these inward-facing conformations of vSGLT show that ions are released quickly from the proposed binding site (28,(32)(33)(34)(35).…”

supporting

confidence: 65%

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: 65%

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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“…Homologous PutP Can Bind Two Substrate Molecules-To test whether other SSS members also exhibit an MBS larger than unity, we performed equilibrium dialysis-based saturation binding of [ 3 H]proline to purified PutP, a well characterized and experimentally tractable SSS (31,38,(43)(44)(45)(46), which has a sequence homology of 42% (sequence identity of 20%) to vSGLT in the TM1Ј-10Ј (Fig. 1).…”

Section: Mutations Of the S C Or S E Site Residues Of Vsglt Reduce Thmentioning

confidence: 99%

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters

Zheng

Lee

Bracher

et al. 2015

Journal of Biological Chemistry

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Background: Although 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. Results: We identified second substrate sites in two SSSs that align with the crystallographically identified site in LeuT. Conclusion: Substrate transport by SSSs involves two substrate sites. Significance: NSS and SSS share common mechanistic features.

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“…The NIS residues corresponding to these LeuT residues are S353 and T354 (37). In different transporters, individual or double replacement of the β-OH-residues by Ala at the Na2 site can have markedly different consequences (37,43,45,48,(54)(55)(56). Binding of any of the ions to empty NIS increases the affinity of the protein for the other ions (12).…”

Section: Discussionmentioning

confidence: 99%

Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter

Ferrandino

Nicola

Sánchez

et al. 2016

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

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The sodium/iodide symporter (NIS) mediates active I− transport in the thyroid—the first step in thyroid hormone biosynthesis—with a 2 Na+: 1 I− stoichiometry. The two Na+ binding sites (Na1 and Na2) and the I− binding site interact allosterically: when Na+ binds to a Na+ site, the affinity of NIS for the other Na+ and for I− increases significantly. In all Na+-dependent transporters with the same fold as NIS, the side chains of two residues, S353 and T354 (NIS numbering), were identified as the Na+ ligands at Na2. To understand the cooperativity between the substrates, we investigated the coordination at the Na2 site. We determined that four other residues—S66, D191, Q194, and Q263—are also involved in Na+ coordination at this site. Experiments in whole cells demonstrated that these four residues participate in transport by NIS: mutations at these positions result in proteins that, although expressed at the plasma membrane, transport little or no I−. These residues are conserved throughout the entire SLC5 family, to which NIS belongs, suggesting that they serve a similar function in the other transporters. Our findings also suggest that the increase in affinity that each site displays when an ion binds to another site may result from changes in the dynamics of the transporter. These mechanistic insights deepen our understanding not only of NIS but also of other transporters, including many that, like NIS, are of great medical relevance.

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Role of Ser-340 and Thr-341 in Transmembrane Domain IX of the Na+/Proline Transporter PutP of Escherichia coli in Ligand Binding and Transport

Cited by 26 publications

References 39 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

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters

Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter

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Role of Ser-340 and Thr-341 in Transmembrane Domain IX of the Na+/Proline Transporter PutP of Escherichia coli in Ligand Binding and Transport

Cited by 26 publications

References 39 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

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters

Na + coordination at the Na2 site of the Na + /I − symporter

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Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter