Nature
09August2007Volume448Number7154pp623726neurotransmittertransporters
LeuT
Nature 09 August 2007 Volume 448 Number 7154, pp623-726 Advance Online
Publication LeuT 2A65
Usage of Human Famsbase for Nature 09 August 2007 Volume 448 Number 7154,
pp623-726 neurotransmitter transporters on Advance Online Publication LeuT 2A65
Advance Online Publication
Antidepressant binding site in a bacterial homologue of neurotransmitter transporters
Satinder K. Singh, Atsuko Yamashita & Eric Gouaux
Coordinates and structure factors for the alanine?sodium?clomipramine and the
leucine?sodium?clomipramine, ?
imipramine and ?desipramine complexes have been deposited in the Protein Data Bank
under accession codes 2QEI, 2Q6H, 2Q72 and 2QB4, respectively.
Status Search : Structure ID=2QEI
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Status codes:
PROC: to be processed; WAIT:
processing started, waiting for author input to continue processing; HPUB: hold until
publication; HOLD: hold until a certain date; WDRN: deposition withdrawn; AUTH:
processed, waiting for author review and approval; POLC: waiting for a policy decision;
REPL: author sent new coordinates, entry to be reprocessed
2QEI
Crystal structure analysis of LeuT complexed with L-alanine, sodium, and
clomipramine
Deposition Date:
2007-06-25
Release Date:
n/a
Exp. Data:
Structure
Factors Deposited
Sequence Available
Status
Authors
NO
PROC
Singh, S.K., Yamashita, A., Gouaux, E.
LeuT is a stable, sodium-coupled leucine transporter from the eubacterium Aquifex
aeolicus and is the only member of the neurotransmitter sodium symporter (NSS or
SLC6) family of secondary transporters that has so far been amenable to structural
analysis17. Eukaryotic NSS counterparts include those that pump neurotransmitters
such as serotonin, norepinephrine, dopamine, glycine and -aminobutyric acid (GABA)
from the synapse to neuronal and glial cytoplasms, shaping the magnitude and duration
of synaptic signalling18. The crystal structure of LeuT provided the first molecular
glimpse into an NSS member17, but it did not yield any clues as to the atomic basis of
inhibition. Motivated by this dearth of knowledge, together with the key role of NSS
proteins and their antagonists in synaptic transmission, we sought to find inhibitors of
LeuT and to characterize their mechanism of action by using flux, binding and
crystallographic methods.We began by screening a wide variety of NSS inhibitors,
including those that target the transporters of glycine (GlyT), GABA (GAT), dopamine
(DAT), norepinephrine (NET), and serotonin (SERT), for their ability to inhibit the
uptake of L-[3H]Leu by purified LeuT reconstituted into lipid vesicles. The tricyclic
antidepressant (TCA) clomipramine was the most potent inhibitor of uptake (Fig. 1a),
whereas the structurally related compounds, imipramine and desipramine, were less
effective (Fig. 1a, Supplementary Fig. 1a). In dose?response experiments (Fig. 1b),
clomipramine was a modestly potent inhibitor of leucine transport, with a half-maximal
inhibitory concentration (IC50) approximately eightfold lower than that of imipramine
(250 versus 2,090 M; Supplementary Table 1). To test whether the TCAs could displace
bound
leucine,
we
conducted
radioligand
binding
experiments
using
detergent-solubilized LeuT, [3H]Leu, and potential cold competitors. Unlike amino acids
such as leucine, alanine and tryptophan, none of the TCAs (at 1 mM) significantly
displaced [3H]Leu from LeuT (Supplementary
Fig. 1b), tentatively excluding competitive inhibition as the mechanism by which the
TCAs inhibit LeuT.
Structure Determination
Diffraction data were collected at 110K at NSLS beamlines X26C (20-sec
exposures) and X29A (5-sec exposures) or ALS beamline 8.2.2 (3-sec exposures) at
an X-ray wavelength of 1.1 or 1.0 Å in a 180º or 360° sweep with 1.0º oscillation
doi: 10.1038/nature06038 SUPPLEMENTARY INFORMATION
www.nature.com/nature 10
and processed with HKL200031. All four inhibitor complex crystals diffracted to
beyond 2.0Å resolution and were indexed in the space group C2. Unit cell
dimensions varied slightly from crystal to crystal, but all were approximately
a=88.0-89.8 Å, b=86.0-86.8 Å, c=80.8-81.7 Å, β=95.9-96.9º. Phases for the CMI
complex were obtained via difference Fourier techniques employing the original
LeuT structure (PDB ID 2A65)16 as the starting model. Subsequent model building
was accomplished in O32 with the assistance of sigma-weighted 2Fo-Fc and Fo-Fc
maps as well as simulated-annealing Fo-Fc omit maps. Refinement was performed
with CNS33. This sequence was repeated reiteratively until the Rfactor and Rfree
values converged, at which point L-leucine or L-alanine, two sodium ions, n-octylβ-D-glucopyranoside, CMI34, and water molecules were added. Refinement then
progressed until Rfactor and Rfree converged again. Once the LeuT-leucine-CMI
structure was completed, it was subsequently used to procure phases for the
LeuTalanineCMI and LeuT-leucine-IMI35 complexes, the latter of which was then
employed for the desipramine complex. For all four structures, Ramachandran
geometry is excellent, with greater than 93% of the residues in the most favored
regions and none in disallowed regions. For each data set, the test reflections (4.9%
of the total) were selected so that they coincided with those employed in the original
structure determination (PDB ID 2A65)16, which were chosen randomly.
The Poisson-Boltzman equation for electrostatic calculations was solved
according to the algorithm implemented in the APBS module36, 37 of PyMOL, and all
structure figures were generated with PyMol38.
doi: 10.1038/nature06038 SUPPLEMENTARY INFORMATION
www.nature.com/nature 11
Supplementary Notes (Literature Cited)
31. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in
oscillation mode. Methods Enzymol. 276, 307-326 (1997).
32. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for
building protein models in electron density maps and the location of errors in
these models. Acta Crystallogr. A 47, 110-119 (1991).
33. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for
macromolecular structure determination. Acta Crystallogr. D 54, 905-921 (1998).
34. Post, M. L. & Horn, A. S. The crystal and molecular structure of the tricyclic
antidepressant chlorimipramine hydrochloride: 3-Chloro-5-(3dimethylaminopropyl)-10,11-dihydro-5H-dibenz[b,f]azepine hydrochloride. Acta
Crystallogr. B 33, 2590-2595 (1977).
35. Post, M. L., Kennard, O., & Horn, A.S. The Tricyclic antidepressants: imipramine
hydrochloride. The Crystal and molecular structure of 5-(3dimethylaminopropyl)-10,11-dihydro-5H-dibenz[b,f]azepine hydrochloride. Acta
Crystallogr. B 31, 1008-1013 (1975).
36. Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics
of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad.
Sci. USA 98, 10037-10041 (2001).
37. Lerner, M. G. & Carlson., H. A. (University of Michigan, Ann Arbor, 2006).
38. DeLano, W. L. (DeLano Scientific, San Carlos, CA, 2002).
doi: 10.1038/nature06038 SUPPLEMENTARY INFORMATION
www.nature.com/nature 12
Na(+):neurotransmitter symporter (Snf family) (2A65:A)
2A65
Title
Authors
Crystal structure of LEUTAA, a bacterial homolog of
Na+/Cl--dependent neurotransmitter transporters
Yamashita, A., Singh, S.K., Kawate, T., Jin, Y., Gouaux, E.
Yamashita, A., Singh, S.K., Kawate, T., Jin, Y., Gouaux, E. Crystal
Primary
Citation
structure of a bacterial homologue of Na(+)/Cl(-)-dependent
neurotransmitter transporters. Nature v437 pp. 215-223, 2005
[ Abstract ]
History Deposition
Experimental
Method
Parameters
Unit Cell
Type
2005-07-01 Release 2005-08-02
X-RAY DIFFRACTION Data
[ EDS ]
Resolution[Å]
R-Value
R-Free
Space Group
1.65
0.199 (work)
0.217
C 2 (C 1 2 1)
Length [Å]
a
87.86
b
86.31
c
81.02
Angles [°]
alpha
90.00
beta
95.73
gamma
90.00
Molecular
Description Polymer: 1
Asymmetric family)
Molecule: Na(+):neurotransmitter symporter (Snf
Chains: A
Unit
Classification Transport Protein
Source Polymer: 1
Scientific Name: Aquifex aeolicus vf5
Expression
system: Escherichia coli
Ligand Chemical
Component Identifier Name
Formula
Drug
Hapten
Ligand
Similarity Similarity Structure
Ligand
Interaction
BOG
B-OCTYLGLUCOSIDE
CL
CHLORIDE ION
LEU
LEUCINE
NA
SODIUM ION
GO Terms Polymer
C14 H28
[ View ] [ View ]
O6
[ View ] [ View ]
Cl
C6 H13 N
[ View ] [ View ]
O2
[ View ] [ View ]
Na
Molecular Function
Biological Process
Cellular Component
Na(+):neurotransmitter
symporter (Snf family)

none
(2A65:A)
neurotransmitter symporter (Snf family)
Na(+):neurotransmitter symporter (Snf family) (2A65:A)

none

none
>2A65:A|PDBID|CHAIN|SEQUENCE
MEVKREHWATRLGLILAMAGNAVGLGNFLRFPVQAAENGGGAFMIPYIIAFLLVGIP
LMWIEWAMGRYGGAQGHGTTPAI
FYLLWRNRFAKILGVFGLWIPLVVAIYYVYIESWTLGFAIKFLVGLVPEPPPNATDPDS
ILRPFKEFLYSYIGVPKGDEP
ILKPSLFAYIVFLITMFINVSILIRGISKGIERFAKIAMPTLFILAVFLVIRVFLLETPNGT
AADGLNFLWTPDFEKLKD
PGVWIAAVGQIFFTLSLGFGAIITYASYVRKDQDIVLSGLTAATLNEKAEVILGGSISIP
AAVAFFGVANAVAIAKAGAF
NLGFITLPAIFSQTAGGTFLGFLWFFLLFFAGLTSSIAIMQPMIAFLEDELKLSRKHAV
LWTAAIVFFSAHLVMFLNKSL
DEMDFWAGTIGVVFFGLTELIIFFWIFGADKAWEEINRGGIIKVPRIYYYVMRYITPA
FLAVLLVVWAREYIPKIMEETH
WTVWITRFYIIGLFLFLTFLVFLAERRRNHESAGTLVPR
Article Title
Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter
transporters.
Abstract
Na+/Cl--dependent
transporters
terminate
synaptic
transmission
by
using
electrochemical gradients to drive the uptake of neurotransmitters, including the
biogenic amines, from the synapse to the cytoplasm of neurons and glia. These
transporters are the targets of therapeutic and illicit compounds, and their dysfunction
has been implicated in multiple diseases of the nervous system. Here we present the
crystal structure of a bacterial homologue of these transporters from Aquifex aeolicus,
in complex with its substrate, leucine, and two sodium ions. The protein core consists of
the first ten of twelve transmembrane segments, with segments 1-5 related to 6-10 by a
pseudo-two-fold axis in the membrane plane. Leucine and the sodium ions are bound
within the protein core, halfway across the membrane bilayer, in an occluded site devoid
of water. The leucine and ion binding sites are defined by partially unwound
transmembrane helices, with main-chain atoms and helix dipoles having key roles in
substrate and ion binding. The structure reveals the architecture of this important class
of transporter, illuminates the determinants of substrate binding and ion selectivity,
and defines the external and internal gates.
Keywords
Amino Acid Sequence, Bacteria, Bacterial Proteins, Binding Sites, Biological Transport,
Chlorides, Crystallography, X-Ray, Hydrophobicity, Leucine, Membrane Transport
Proteins, Models, Molecular, Molecular Sequence Data, Neurotransmitter Agents,
Sequence Alignment, Sodium, Structure-Activity Relationship, Water
Na(+):neurotransmitter symporter (Snf family) Aquifex aeolicus
Authors
Yamashita, A., Singh, S.K., Kawate, T., Jin, Y., Gouaux, E.
Organizational Affiliation
Department of Biochemistry and Molecular Biophysics and.
Journal
Nature v437 pp. 215-23, 2005
Pubmed ID