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

Khafizov, Kamil and Perez, Camilo and Koshy, Caroline and Quick, Matthias and Fendler, Klaus and Ziegler, Christine and Forrest, Lucy R.. (2012) Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP. Proceedings of the National Academy of Sciences, 109 (44). E3035-E3044.

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Official URL: http://edoc.unibas.ch/56044/

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Abstract

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 sodium-binding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuT-like (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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics
UniBasel Contributors:Perez, Camilo
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:National Academy of Sciences
ISSN:0027-8424
e-ISSN:1091-6490
Note:Publication type according to Uni Basel Research Database: Journal article
Identification Number:
Last Modified:30 Nov 2017 11:19
Deposited On:30 Nov 2017 11:19

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