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PDBsum entry 3f4j
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Transport protein
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PDB id
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3f4j
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References listed in PDB file
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Key reference
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Title
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A competitive inhibitor traps leut in an open-To-Out conformation.
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Authors
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S.K.Singh,
C.L.Piscitelli,
A.Yamashita,
E.Gouaux.
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Ref.
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Science, 2008,
322,
1655-1661.
[DOI no: ]
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PubMed id
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Note: In the PDB file this reference is
annotated as "TO BE PUBLISHED". The citation details given above have
been manually determined.
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Abstract
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Secondary transporters are workhorses of cellular membranes, catalyzing the
movement of small molecules and ions across the bilayer and coupling substrate
passage to ion gradients. However, the conformational changes that accompany
substrate transport, the mechanism by which a substrate moves through the
transporter, and principles of competitive inhibition remain unclear. We used
crystallographic and functional studies on the leucine transporter (LeuT), a
model for neurotransmitter sodium symporters, to show that various amino acid
substrates induce the same occluded conformational state and that a competitive
inhibitor, tryptophan (Trp), traps LeuT in an open-to-out conformation. In the
Trp complex, the extracellular gate residues arginine 30 and aspartic acid 404
define a second weak binding site for substrates or inhibitors as they permeate
from the extracellular solution to the primary substrate site, which
demonstrates how residues that participate in gating also mediate permeation.
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Figure 4.
Fig. 4. A second Trp molecule is bound between R30 and D404 of
the extracellular gate only in the open-to-out conformation. (A)
Trp602 bound in the extracellular vestibule of LeuT, residing
between D404 and R30, flanked by the  -helix in TM10. (B)
Extracellular vestibule of the LeuT-SeMet complex.
Anomalous-difference Fourier map (contoured at 5  and 15 and depicted in
green and blue mesh, respectively) showing no substantial
density peaks in the extracellular vestibule.
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Figure 5.
Fig. 5. Schematic of transport and inhibition in LeuT.
Postulated conformational changes associated with isomerization
from the open-to-out (A) to the outward-facing occluded state
(B) on binding of substrate and ions, from the occluded (B) to
open-to-in state (C) and dissociation of transported substrate
and ions, and from the open-to-in (C) back to the open-to-out
state (A). (D) Effect of the competitive inhibitor Trp on
transport: stabilizing the open-to-out conformation. (E) Effect
of the noncompetitive TCA inhibitors on transport-stabilizing
the outward-facing occluded conformation. The boxed
conformations represent actual crystal structures, whereas the
unboxed conformations are hypothetical.
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The above figures are
reprinted
by permission from the AAAs:
Science
(2008,
322,
1655-1661)
copyright 2008.
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Secondary reference #1
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Title
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Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.
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Authors
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S.K.Singh,
A.Yamashita,
E.Gouaux.
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Ref.
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Nature, 2007,
448,
952-956.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2: TCAs bind in the putative permeation pathway of LeuT.
F[o]–F[c] simulated annealing omit maps, both contoured at
3.0  ,
of clomipramine (a) and imipramine (b), in which the TCA was
omitted from the simulated annealing run and subsequent phase
calculation. The chlorine atom of clomipramine is green. c, LeuT
tilted  15°
from the membrane plane to illustrate the binding sites of
clomipramine and leucine, both depicted in space-filling
representation. Helices whose residues interact with
clomipramine are coloured. d, Electrostatic properties of the
LeuT extracellular-facing vestibule, with clomipramine
(yellow) cradled in the negatively charged crevice.
Electrostatic potential isocontours were set at +7 kT e^–1
(blue) and –7 kT e^–1 (red). EL4 is tinted green. To make
clomipramine more visible, LeuT has been tilted an additional
5°
towards the reader from the view in c ( 20°
from the membrane plane).
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Figure 3.
Figure 3: Clomipramine-binding site. a, Stereo view, with
clomipramine and leucine carbon atoms depicted in yellow, sodium
ions in purple and two water molecules in red. Residues whose
interactions with clomipramine are hydrophobic, polar (Q34) or
ionic (D401) are coloured grey, orange and pink, respectively.
Atoms depicted as spheres interact through either a hydrogen
bond or a salt bridge. b, F[o]–F[c] simulated annealing omit
map, contoured at 3.0 ,
of D404 and R30, depicting the direct salt bridge between the
guanidium of R30 and the carboxylate of D404, with displacement
of two water molecules (compare to overlay in d). Distances
(Å) are shown along dashed lines. c, F[o]–F[c] simulated
annealing omit map, contoured at 3.0 ,
of the tip of EL4 (residues A317–N321), illustrating the
movement 'up' of A319 in the LeuT–clomipramine crystal
structure (compare to overlay in d). d, Same view as in a with
residues from the original LeuT structure (PDB ID 2A65) overlaid
(in green with two water molecules between D404 and R30 in cyan)
onto those from the LeuT–clomipramine crystal structure (in
pink with the two displaced waters in red and labelled as H[2]O
(CMI)). e, CPK rendering of clomipramine, R30 and F253
illustrating how the positively charged guanidium group is
sandwiched between the aromatic rings of clomipramine and F253.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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Secondary reference #2
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Title
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Crystal structure of a bacterial homologue of na+/cl--Dependent neurotransmitter transporters.
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Authors
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A.Yamashita,
S.K.Singh,
T.Kawate,
Y.Jin,
E.Gouaux.
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Ref.
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Nature, 2005,
437,
215-223.
[DOI no: ]
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PubMed id
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Figure 5.
Figure 5: Extracellular and cytoplasmic gates. a, Slice
through the surface of LeuT[Aa], viewed parallel to the
membrane, showing the extracellular cavity. Connolly surface of
LeuT[Aa] is shown in beige. l-Leucine, Tyr 108, Phe 253 and the
two charged pairs (Arg 30 -Asp 404 and Arg 5 -Asp 369) are
depicted as stick models in yellow for leucine, purple for
aromatic residues, blue for arginines and red for aspartates. b,
c, Key interacting residues at the extracellular (b) and at the
cytoplasmic (c) gate.
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Figure 6.
Figure 6: Speculative transport mechanism. Schematic drawing
of a possible conformational change upon substrate/sodium ion
transport. The left panel shows the outward-facing state. TM1a
and TM6b assume the closed arrangement, whereas TM1b and TM6a
adopt the open one. The middle panel shows the
substrate-occluded state, which corresponds to the current
crystal structure. TM1a and TM6b assume the closed arrangement,
whereas TM1b and TM6a adopt a partially open one with some
residues blocking the permeation pathway. The right panel shows
the inward-facing state. TM1b and TM6a assume the closed
arrangement, whereas TM1a and TM6b adopt the closed one, to open
the pathway to the cytoplasm.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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