| Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP. | |
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MedLine Citation:
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PMID: 23047697 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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 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. |
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Authors:
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Kamil Khafizov; Camilo Perez; Caroline Koshy; Matthias Quick; Klaus Fendler; Christine Ziegler; Lucy R Forrest |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2012-10-09 |
Journal Detail:
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Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 109 ISSN: 1091-6490 ISO Abbreviation: Proc. Natl. Acad. Sci. U.S.A. Publication Date: 2012 Oct |
Date Detail:
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Created Date: 2012-11-01 Completed Date: 2013-01-08 Revised Date: 2013-04-30 |
Medline Journal Info:
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Nlm Unique ID: 7505876 Medline TA: Proc Natl Acad Sci U S A Country: United States |
Other Details:
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Languages: eng Pagination: E3035-44 Citation Subset: IM |
Affiliation:
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Computational Structural Biology Group, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Binding Sites Carrier Proteins / chemistry, genetics, metabolism* Crystallography, X-Ray Models, Molecular Molecular Dynamics Simulation Molecular Sequence Data Mutagenesis, Site-Directed Sequence Homology, Amino Acid Sodium / metabolism* |
| Chemical | |
Reg. No./Substance:
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0/Carrier Proteins; 146313-33-9/betaine plasma membrane transport proteins; 7440-23-5/Sodium |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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