Abstract
LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 861-866 |
| Number of pages | 6 |
| Journal | EMBO Reports |
| Volume | 13 |
| Issue number | 9 |
| DOIs | |
| State | Published - Sep 2012 |
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Keywords
- neurotransmitter uptake
- sodium-coupled transporter
- X-ray crystallography
ASJC Scopus subject areas
- Genetics
- Molecular Biology
- Biochemistry
Cite this
Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue. / Wang, Hui; Gouaux, Eric.
In: EMBO Reports, Vol. 13, No. 9, 09.2012, p. 861-866.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue
AU - Wang, Hui
AU - Gouaux, Eric
PY - 2012/9
Y1 - 2012/9
N2 - LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.
AB - LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.
KW - neurotransmitter uptake
KW - sodium-coupled transporter
KW - X-ray crystallography
UR - http://www.scopus.com/inward/record.url?scp=84865730840&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84865730840&partnerID=8YFLogxK
U2 - 10.1038/embor.2012.110
DO - 10.1038/embor.2012.110
M3 - Article
C2 - 22836580
AN - SCOPUS:84865730840
VL - 13
SP - 861
EP - 866
JO - EMBO Reports
JF - EMBO Reports
SN - 1469-221X
IS - 9
ER -