Document Detail


Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP.
MedLine Citation:
PMID:  23047697     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Kamil Khafizov; Camilo Perez; Caroline Koshy; Matthias Quick; Klaus Fendler; Christine Ziegler; Lucy R Forrest
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-10-09
Journal Detail:
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:
Created Date:  2012-11-01     Completed Date:  2013-01-08     Revised Date:  2013-07-11    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  E3035-44     Citation Subset:  IM    
Affiliation:
Computational Structural Biology Group, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany.
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MeSH Terms
Descriptor/Qualifier:
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:
0/Carrier Proteins; 146313-33-9/betaine plasma membrane transport proteins; 7440-23-5/Sodium
Comments/Corrections

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