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  Investigation of the sodium binding sites in the betaine transporter BetP

Khafizov, K., Perez, C., Koshy, C., Quick, M., Fendler, K., Ziegler, C., et al. (2012). Investigation of the sodium binding sites in the betaine transporter BetP. Proceedings of the National Academy of Sciences of the United States of America, 109(44), E3035-E3045. doi:10.1073/pnas.1209039109.

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 Creators:
Khafizov, Kamil1, Author           
Perez, Camilo2, Author           
Koshy, Caroline2, Author           
Quick, Matthias3, Author
Fendler, Klaus4, Author           
Ziegler, Christine2, Author           
Forrest, Lucy R.1, Author           
Affiliations:
1Max Planck Research Group of Computational Structural Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068293              
2Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068291              
3External Organizations, ou_persistent22              
4Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society, ou_2068289              

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Free keywords: secondary transport; symmetry; membrane protein; alkali metal ion; osmoregulation
 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 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 Km and Kd for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22Na+ 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.

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Language(s): eng - English
 Dates: 2012-10-092012-10-30
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 631071
DOI: 10.1073/pnas.1209039109
 Degree: -

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Title: Proceedings of the National Academy of Sciences of the United States of America
  Other : PNAS
  Other : Proceedings of the National Academy of Sciences of the USA
  Abbreviation : Proc. Natl. Acad. Sci. U. S. A.
Source Genre: Journal
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Publ. Info: Washington, D.C. : National Academy of Sciences
Pages: - Volume / Issue: 109 (44) Sequence Number: - Start / End Page: E3035 - E3045 Identifier: ISSN: 0027-8424
CoNE: https://pure.mpg.de/cone/journals/resource/954925427230