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 OF THE UNITED STATES OF AMERICA, 109 (44). E3035-E3044. ISSN 0027-8424
Full text not available from this repository. (Request a copy)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 Na-22(+) 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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | TRANSMEMBRANE DOMAIN-IX; CRYSTAL-STRUCTURE; MOLECULAR-DYNAMICS; ESCHERICHIA-COLI; GLUTAMATE TRANSPORTERS; MEMBRANE-TRANSPORT; ALTERNATING ACCESS; BACTERIAL HOMOLOG; STRUCTURAL BASIS; CARRIER BETP; secondary transport; symmetry; membrane protein; alkali metal ion; osmoregulation |
| Subjects: | 500 Science > 540 Chemistry & allied sciences 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Christine Ziegler |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 01 Jun 2022 05:25 |
| Last Modified: | 01 Jun 2022 05:25 |
| URI: | https://pred.uni-regensburg.de/id/eprint/17922 |
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