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Zhou et al.
Fig. 1. a. Overall structure of the LeuT-sertraline complex viewed from within the
membrane plane. The SSRI molecule binds LeuT between the tips of the EL4 hairpin
loop and the extracellular gate formed by residues Arg30, Tyr108, Phe253 and Asp404.
Both the R- and S-fluoxetines bind at the same position, as do the TCA molecules. The
substrate leucine is colored red, the gate residues purple, and sertraline grey. b. Spatial
relationship between the drug-binding site and the substrate-binding site. The sertralinebinding site and the leucine-binding site share four common amino acid residues,
Leu25, G26, Y108 and F253, even though the two binding sites do not share common
physical space. Dashed lines represent hydrogen bonds.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602
Fig. 2. Surface representation of the halogen-binding pocket in a, LeuT-sertraline
complex and, b, LeuT-R-fluoxetine complex.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602
Fig. 3. Superposition and comparison of three SSRI structures in their LeuTbound form and free form, the latter previously determined using small molecule
crystallography 1,2. a, Sertraline, b, R-fluoxetine and, c, S-fluoxetine. For each SSRI,
the main change is a rotation round the bond that connects the halogen-substituted
phenyl ring and the rest moiety of the molecule. The torsion angle changes by 180˚ ( of
C5-C4-C13-C14), 46˚ (of C3-O5-C6-C7) and 19˚ (of C3-O5-C6-C7), for sertraline, Rfluoxetine and S-fluoxetine, respectively. The free forms are colored in grey, and LeuTbound forms are colored yellow, orange and green, respectively.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602
Fig. 4. Superposition of LeuT structures when complexed with sertraline, Rfluoxetine and S-fluoxetine, respectively. The drug molecules are removed for clarity.
The protein is colored yellow when bound to sertraline, orange when bound to Rfluoxetine, and green when bound to S-fluoxetine.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602
Fig. 5. Curves showing affinities for antidepressant of human SERT, NET and
DAT as measured using binding assays in HEK293 cells, when a residue at their HBP is
mutated. The appropriate wild-type transporter was compared. a - c. Binding of
[3H]citalopram to the SERT-Ile179Asp mutant in the presence of sertraline,
chlomipramine and desipramine. d - f. Binding of [3H]CFT to the NET-Ala77Gly mutant
and to the DAT-Ala81Gly mutant (g - i) both in the presence of sertraline, R-fluoxetine
and S-fluoxetine. Data points are expressed as percent of specific binding in the
absence of antidepressant, and are the mean + S.E.M. of three to five experiments.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602
Fig. 6. Superposition and comparison of SSRIs and TCAs bound to LeuT. a,
Superposition of structures of LeuT-S-fluoxetine and LeuT-desipramine. The amine
groups from both S-fluoxetine (SFXT) and desipramine (DSM) are located at the same
position, and their amine nitrogen atoms interact with Asp401. The S-fluoxetine
molecule is colored green, and the desipramine molecule grey. b, Superposition of
structures of LeuT-S-fluoxetine and LeuT-clomipramine. The chloride atom from
clomipramine (CMI) does not insert into the same halogen-binding pocket as the SSRIs
do. The S-fluoxetine molecule is colored green, and the chloripramine molecule purple
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602
Fig. 7. Sequence alignment between NSS proteins at the EL4 hairpin loop. The
arrow indicates an alanine insertion in the SERT sequence.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602
Table 1. Affinity measurements showing the effect of mutations in the halogen-binding
pocket on inhibition of [3H]radioligand binding to SERT-, NET- and DAT-expressing
HEK293 cells
[3H]Citalopram binding to SERT constructs
IC50 (nM)
Sertraline
Clomipramine
Desipramine
SERT-WT
4.22±1.37
7.01±1.44
25.0±6.0
SERT-Ile179Asp
290±68*
7.10±1.18
15.4±4.0
[3H]CFT binding to NET constructs
IC50 (nM)
Sertraline
R-fluoxetine
S-fluoxetine
NET-WT
474±60
193±18
363±43
NET-Ala77Gly
262±17**
99.6±14.8*
159±13**
[3H]CFT binding to DAT constructs
IC50 (nM)
Sertraline
R-fluoxetine
S-fluoxetine
DAT-WT
156±34
396±33
1653±331
DAT-Ala81Gly
41.1±7.3*
170±31*
573±63*
Notes: Values are the mean ± S.E.M. of three to five experiments. The significance of
measurements is indicated by * (P < 0.05) and ** (P < 0.005) compared with wild-type
(Student’s t-test).
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602
References:
1.
2.
Caruso, F., Besmer, A. & Rossi, M. The absolute configuration of sertraline
(Zoloft) hydrochloride. Acta Cryst. C55, 1712-1714 (1999).
Robertson, D.W., Jones, N.D., Swartzendruber, J.K., Yang, K.S. & Wong, D.T.
Molecular structure of fluoxetine hydrochloride, a highly selective serotoninuptake inhibitor. J Med Chem 31, 185-9 (1988).
Nature Structural & Molecular Biology: doi:10.1038/nsmb.1602