Three-Dimensional Structure of TspO by Electron Cryomicroscopy of Helical Crystals
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Transcript of Three-Dimensional Structure of TspO by Electron Cryomicroscopy of Helical Crystals
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Structure
Article
Three-Dimensional Structure of TspOby Electron Cryomicroscopy of Helical CrystalsVladimir M. Korkhov,1,2,3,* Carsten Sachse,1,2,4 Judith M. Short,1 and Christopher G. Tate1,*1Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK2These authors contributed equally to this work3Present address: Institute of Molecular Biology and Biophysics, Eidgenossische Technische Hochschule Hoenggerberg, Schafmattstrasse20, HPK D11, 8046 Zurich, Switzerland4Present address: European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
*Correspondence: [email protected] (V.M.K.), [email protected] (C.G.T.)
DOI 10.1016/j.str.2010.03.001
SUMMARY
The 18 kDa TSPO protein is a polytopic mitochondrialouter membrane protein involved in a wide range ofphysiological functions and pathologies, includingneurodegeneration and cancer. The pharmacologyof TSPO has been extensively studied, but little isknown about its biochemistry, oligomeric state, andstructure. We have expressed, purified, and charac-terized a homologous protein, TspO from Rhodo-bacter sphaeroides, and reconstituted it as helicalcrystals. Using electron cryomicroscopy and single-particle helical reconstruction, we have determineda three-dimensional structure of TspO at 10 A resolu-tion. The structure suggests that monomeric TspOcomprises five transmembrane a helices that forma homodimer, which is consistent with the dimericstate observed in detergent solution. Furthermore,the arrangement of transmembrane domains of indi-vidual TspO subunits indicates a possibility of twosubstrate translocation pathways per dimer. Thestructure provides the first insight into the moleculararchitecture of TSPO/PBR protein family that willserve as a framework for future studies.
INTRODUCTION
The human 18 kDa translocator protein (TSPO), formerly known
as the peripheral benzodiazepine receptor (Papadopoulos et al.,
2006a), is involved in a wide range of physiological functions
and pathological conditions. TSPO is also among the key targets
of Valium (diazepam), which is one of the most frequently
prescribed drugs for anxiety disorders (Braestrup et al., 1977;
Gavish et al., 1999). Among the established molecular functions
of TSPO are translocation of cholesterol (Li and Papadopoulos,
1998; Li et al., 2001) and porphyrins (Taketani et al., 1994) across
the mitochondrial outer membrane, where a large fraction of
TSPO is normally localized within the cell. On a cellular level,
TSPO has been shown to be involved in steroid biosynthesis
(Lacapere and Papadopoulos, 2003), cellular respiration (O’Hara
et al., 2003), proliferation (Galiegue et al., 2004), and apoptosis
Structure 18
(Maaser et al., 2001). In addition, biochemical and pharmacolog-
ical data have implicated TSPO in a wide range of pathological
conditions, including epilepsy (Veenman et al., 2002), neurode-
generative diseases (Papadopoulos et al., 2006b), and cancer
(Han et al., 2003; Maaser et al., 2002; Weisinger et al., 2004).
TSPO is also an established positron emission tomography
marker for pathologies of the central nervous system (Zhang
et al., 2004). A wealth of pharmacological data on TSPO has
been published and high-affinity ligands have been developed
(Papadopoulos et al., 2006a; Scarf et al., 2009). However,
although attempts have been made to study the purified and
reconstituted TSPO (Delavoie et al., 2003; Garnier et al., 1994;
Lacapere et al., 2001), little is known about its three-dimensional
(3D) structure, oligomeric state, and mode of action, i.e., whether
it operates as a pump, a transporter, or a channel (Papadopoulos
et al., 2006a). Bioinformatic predictions and hydropathy anal-
yses, confirmed by biochemical and biophysical evidence,
have indicated that TSPO and its bacterial homologs probably
contain five a-helical transmembrane domains that possibly
form dimers or multimers (Bogan et al., 2007; Papadopoulos
et al., 1994; Yeliseev and Kaplan, 2000).
Structural studies of eukaryotic membrane proteins are still
difficult. Expressing, purifying, and stabilizing eukaryotic mem-
brane proteins presents formidable obstacles that need to be
overcome before their structures can be determined by X-ray
crystallography, electron cryomicroscopy (cryo-EM), or nuclear
magnetic resonance (NMR). To obtain insight into the molecular
structure of the proteins belonging to the TSPO family, we have
therefore used a bacterial homolog, tryptophan-rich sensory
protein (TspO) from Rhodobacter sphaeroides, as a structural
and functional model of the human protein.
TspO from R. sphaeroides is involved in the control of photo-
synthetic gene expression and it has been proposed to act as
an oxygen sensor (Yeliseev et al., 1997) that downregulates the
photopigment genes in response to oxygen (Yeliseev and
Kaplan, 1995, 1999; Zeng and Kaplan, 2001). Its primary struc-
ture is very similar to that of the human TSPO, with 33.5% iden-
tity between the aligned amino acid sequences of R. sphaeroides
TspO and human TSPO (Figure 1), excluding the longer interhel-
ical loops in TSPO (Yeliseev and Kaplan, 1995); this level of
homology is highly significant. Furthermore, bacterial TspO has
been suggested to have functional and structural similarities to
the human protein (Yeliseev and Kaplan, 2000), making it an ideal
candidate for biochemical and structural work. Here we report
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Figure 1. Comparison of Sequence and
Topology of the Human and Bacterial TspO
Homologues
(A) Alignment between the amino acid sequences
of human TSPO (hTSPO) and TspO from R. sphaer-
oides (rsTspO). Bars underneath the sequences
represent hydrophobic regions that are predicted
by hydropathy analysis to form transmembrane
domains.
(B) Cartoon of the predicted topology of R. sphaer-
oides TspO based on hydropathy analysis and the
‘‘positive inside’’ rule.
Structure
Cryo-EM Structure of TspO
the structure of R. sphaeroides TspO at 10 A resolution, which
represents the first structural data for a member of this family.
RESULTS
Expression and Purification of TspO in Escherichia coli
TspO from R. sphaeroides was expressed in E. coli strain
BL21(DE3) (Figure 2A). Contrary to the reports of TspO localiza-
tion in the outer membrane of R. sphaeroides, the recombinantly
expressed protein was inserted predominantly into the E. coli
inner membrane (Figure 2B and 2C). This is unsurprising, given
the predicted helical nature of TspO and the fact that all known
bacterial outer membrane proteins have been shown to be
b-barrel proteins, with the exception of Wza that contains a single
C-terminal transmembrane a helix (Dong et al., 2006). TspO was
expressed with a C-terminal His6 tag, which allowed its purifica-
tion in dodecylmaltoside (DDM) by Ni2+-affinity chromatography
and preparative-scale size exclusion chromatography. The puri-
fied TspO was monodisperse by size exclusion chromatography
(Figure 2F) and was highly pure on Coomassie blue-stained SDS
polyacrylamide gels (Figure 2A).
Detergent-Solubilized TspO Is DimericTspO purified in DDM was subjected to blue native PAGE anal-
ysis (Figures 2D and 2E). TspO formed two bands on the gel,
with the predominant species appearing at an apparent molec-
ular weight of �80 kDa and a minor species at an apparent
678 Structure 18, 677–687, June 9, 2010 ª2010 Elsevier Ltd All rights reserved
molecular weight of �160 kDa. As these
sizes include the mass of bound deter-
gent, lipids, and Coomassie blue, it is
not possible to use these data directly to
determine the oligomeric state of TspO.
However, when TspO was incubated in
1% SDS, the apparent mass decreased,
which is consistent with the disruption of
an oligomeric species by the harsh deter-
gent. For comparison, EmrE was used as
a control because it is known to be a
homodimeric membrane protein (Butler
et al., 2004) of comparable size (15 kDa
in its tagged form) to TspO (18 kDa);
the mobility of TspO in the blue native
gels in the presence or absence of SDS
is virtually identical to EmrE. All these
data are consistent with TspO purified in
DDM being a dimer. The variable appearance of a higher molec-
ular weight band in the TspO sample was likely due to protein
aggregation during its concentration because the higher molec-
ular weight fraction was negligible when the concentration of
purified TspO was kept below 5 mg/ml (Figure 2E). This is consis-
tent with size exclusion chromatography of TspO purified in DDM
showing a monodisperse protein peak (Figure 2F) and is similar
to the behavior of mouse TSPO upon analysis by blue native
PAGE (Rone et al., 2009).
Purified TspO in Detergent Binds PorphyrinsTo establish the functional integrity of the expressed and purified
TspO, we first performed in vivo and in vitro uptake and binding
assays on either intact E. coli or membrane preparations,
respectively, both containing overexpressed TspO. Although
the results of these experiments were indicative of an ability of
the expressed TspO to bind and transport porphyrins, there
was very high background binding to control cells not expressing
TspO, probably because of the hydrophobicity of porphyrins,
precluding accurate determination of substrate transport and
binding affinity constants (data not shown). Therefore, intrinsic
tryptophan fluorescence quenching was used to monitor
porphyrin binding to purified TspO (Figure 3). We tested the
binding of three different porphyrin analogs to purified TspO,
namely, protoporphyrin IX (PPIX), hemin, and coproporphyrin
III (cPPIII). Titration of TspO with increasing ligand concentra-
tions revealed an efficient fluorescence quenching by all three
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Figure 2. Dimeric TspO Is Expressed in the
E. coli Inner Membrane
(A) TspO protein expression was confirmed by
Coomassie blue staining of the SDS-PAGE gel.
TspO was enriched in a small-scale batch NiNTA
purification procedure in DDM (lane TspO) and
compared with control cells (lane pET). DDM-puri-
fied TspO is shown for comparison (lane TspO).
(B and C) Fractionation of the TspO-expressing
BL21(DE3) cell membranes. Lane OM corre-
sponds to the outer membrane; lanes IM1–IM3
correspond to the inner membrane band (IM2)
and the flanking fractions IM1 and IM3 that also
contained inner membrane material. The gel in
(B) was stained with Coomassie blue, whereas
(C) is a western blot of an identical gel probed
with an anti-poly His antibody to identify the posi-
tion of recombinant TspO.
(D and E) Blue native PAGE of purified TspO and
purified EmrE. The asterisk indicates the mono-
meric species formed upon addition of 1% SDS
for 10 min prior to the addition of the loading dye
(+SDS). As a control, a standard dimeric mem-
brane protein of a comparable size (15.2 kDa
including the C-terminal tag), EmrE, is shown.
Two and three asterisks indicate dimeric and mul-
timeric protein species, respectively. If TspO is
concentrated to below 5 mg/ml, the multimeric
band is negligible (E).
(F) Size exclusion chromatography profile of TspO
in DDM.
Structure
Cryo-EM Structure of TspO
analogs (Figures 3A–3C), with apparent half-maximal binding
constants, Kapp, of 8.6 ± 0.6 mM for PPIX, 10.0 ± 2.5 mM for
hemin, and 17.9 ± 1.7 mM for cPPIII (the values are mean ± SD).
All three porphyrin analogs interact with TspO with similar affin-
ities, which suggests that TspO may have a broad range of
porphyrin specificity in vivo. These data confirmed that TspO
was expressed and purified in a functional state. Moreover, the
observed values are comparable to the reported affinity of
PPIX for mouse TSPO, with a Ki of 18.95 mM (Wendler et al.,
2003). No fluorescence quenching was observed when the non-
transported molecule D-aminolevulinic acid was added to TspO
(Figure 3D).
TspO Reconstitution Yields Tubular Helical CrystalsPurified TspO was dialyzed in the presence of purified lipids in
attempts to produce well-ordered 2D crystals for cryo-EM.
A systematic screen of the reconstitution conditions with varying
lipids, lipid-to-protein ratios, buffer composition, temperature,
and dialysis time was performed, which led to conditions where
TspO formed narrow tubular crystals as observed by electron
microscopy of negatively stained specimens (Figure 4A). TspO
tubes were rapidly frozen on holey carbon grids by plunging
them into liquid ethane and cryo-EM images were collected
under low-dose conditions at liquid nitrogen temperature. The
ice-embedded tubes were 100–1000 nm long and 25 nm in
diameter (Figure 4C). The tubes make up 62% of lipid structures
in the sample with another quarter belonging to a group of spher-
ical vesicles. The remaining 13% of lipid structures consisted
of deformed elliptical vesicles that may represent intermediate
stages of partially assembled crystals (Figure 4C, inset).
Structure 18
Three-Dimensional Reconstruction and MolecularArchitecture of TspO in the MembraneTo obtain 3D structural information of TspO, tubes were
analyzed by cryo-EM and a 10 A resolution map was determined
using single-particle helical reconstruction. The micrographs of
well-preserved tubes showed a clearly distinguishable stacked
disk-like morphology (Figure 4D). From 36 micrographs, a total
of 205 straight tubes were segmented into 7542 overlapping
image squares of an 108 nm dimension. Initially, the principal
helical organization of the tubes could be derived from Fourier
analysis of averaged tube images (see Figures S1B and S1C
available online).
To ensure that all the processed tubes consisted of the same
helical packing, we used multivariate statistical analysis to
classify tube segments to carefully analyze their width and the
helical diffraction pattern. First, the width of all individual seg-
ments and their class averages was determined to be 25 nm
(Figure 4E). Second, two out of twelve classes gave rise to an
interpretable set of three identical layer lines (Figure S1B).
Subsequent indexing allowed the building of the helical lattice
(Figures S1C and S1D; see Experimental Procedures). Hence,
the tubes were composed of arrays of stacked rings each
consisting of 12 TspO dimers, each disk being 32.7 A thick
and related to the neighboring disk by a rotation of 9.5� about
the helical axis (Figure S1D). Subsequently, the derived helical
symmetry parameters were refined using single-particle pro-
cessing (Figures S1F and S1G) (Low et al., 2009). For the 3D
image reconstruction, we used a recently developed single
particle-based method that exploits helical symmetry (Sachse
et al., 2007). To further include only those segments with a visible
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Figure 3. Binding of Substrates to TspO
TspO purified in DDM was titrated with increasing amounts of PPIX (A), hemin
(B), and cPPIII (C) and the tryptophan fluorescence was measured at 340 nm.
Structure
Cryo-EM Structure of TspO
680 Structure 18, 677–687, June 9, 2010 ª2010 Elsevier Ltd All rights
stacking order, we excluded two thirds of tube segments that
were devoid of the 32 A layer line diffraction; 2610 segments,
i.e., 20% of the tube images, were included in the final density
map at 10 A resolution (Figures 4D and 4F; Figure S3) (the statis-
tics of the reconstruction are shown in Table 1).
The resulting EM model of TspO revealed a molecular arrange-
ment in which a pair of TspO monomers form a tightly associated
symmetrical dimer of approximate dimensions 40 3 27 A in the
membrane plane and about 40 A perpendicular to the membrane
(Figure 5). The cross-sections through the final density map
perpendicular to the lipid bilayer plane reveal low-density areas
formed within the individual TspO monomers (Figures 5B–5F).
These low-density areas are apparent also in sections parallel
to the plane of the membrane (Figures 5H–5K), which show
that five discrete density peaks comprise one TspO monomer
(circled in Figures 5H–5K), consistent with the prediction from
hydropathy plots that each monomer contains five transmem-
brane helices. Each TspO dimer in the helical crystal is sur-
rounded by a low-density area corresponding to the annular
lipids, which is evident from Figures 5B–5F and 5H–5K. There
are, of course, other possibilities for which a group of five density
peaks in the dimer corresponds to the TspO monomer. Currently
we favor the interpretation depicted in Figures 5H–5K because of
its simplicity and how well it conforms to generally accepted
principles of membrane protein structure. In addition, there
appears to be no discernable density that could represent poten-
tial interhelical loops between the proposed monomers, which is
also consistent with this interpretation.
Interpretation of the Density MapThe low-resolution density map contained a number of rod-
shaped densities that we have interpreted as transmembrane
a helices. We have fitted a-helical backbones manually into these
densities (Figures 6A–6E; Figure S4) either by modeling idealized
a helices directly into the continuous density features or by con-
necting the strongest density elements within the map in a way
that satisfied simple a-helical bundle geometry. At 10 A resolu-
tion, it is not possible to assign the TspO sequence to the posi-
tions in the 3D map, so each putative transmembrane a helix is
labeled a–e in one monomer and a0–e0 in the other monomer of
the TspO dimer (Figure 6A). The interface between the two
TspO monomers is formed by helices a, b, a0, and b0 (Figures
6A and 6D) and it is relatively flat without any large invaginations.
Helices b and b0 of the two parallel monomers are the main struc-
tural elements that appear to be making intermonomer contacts
throughout the lipid bilayer (Figures 6A and 6B). In contrast, a cleft
is formed between helices d and e within the monomer, with helix
e being the most highly tilted of all the helices (Figures 6C and 6E).
Helix c forms contacts predominantly with helices b and d,
For comparison, the measurements using the nontransported molecule
D-aminolevulinic acid (ALA) is shown in (D). The tryptophan fluorescence
emitted at 340 nm was plotted against the concentration of the quenching
reagent to obtain a dose-response curve. The insets in each graph show the
raw spectra in the 300 to 400 nm range. The data shown are the representative
dose-dependence profiles of the tryptophan fluorescence quenching by the
indicated substances; the number of experiments for PPIX, hemin, and cPPIII
were three, two, and two, respectively.
reserved
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Figure 4. Cryo-EM Analysis of the TspO Helical Crystals
(A) An image of negatively stained TspO tubes and small vesicles formed on
TspO reconstitution; the scale bar corresponds to 100 nm.
(B) Cryo-EM of vitrified TspO tubes on a holey carbon grid. The stacked disk
arrangement of the well-preserved tubes is evident from unprocessed images;
the scale bar corresponds to 50 nm.
Table 1. Statistics of Helical Reconstruction
Image Processing Statistics
Resolution FSC at 0.5/0.143 (A) 10.2/7.8
Total length of nonoverlapping segments (nm) 38,894
Number of tubes 205
Number of segments/finally included 7,542/2,610
Segment size (nm) 108
Size of 3D reconstruction (nm) 72
Segment step size (nm) 3.2
Helical rise (A) 32.67
Helical rotation (�) 9.53
Pixel size on the specimen (A) 1.20
Number of asymmetric units 62,640
Structure
Cryo-EM Structure of TspO
Structure 18
although it may possibly form contacts with helix a0 in the adja-
cent monomer.
In addition to the transmembrane densities that we have inter-
preted as a helices, there are large areas of density at the ends of
helices c and e that appear on the outside of the tubes (Figure 6;
Figure S4). This density appears to form the major contact
between adjacent dimers in the crystalline lattice and, in
conjunction with the absence of an equivalent density on the
opposite side of the membrane, it gives the dimer a distinctive
wedge shape that defines the curvature of the tubes and contrib-
utes toward the overall helical crystal geometry. It is unclear at
10 A resolution what the densities at the ends of helices c and
e represent, but it is most likely to be the N terminus and/or C
terminus, the latter still having the His6 tag attached. It seems
less likely that the density represents a loop region between
helices d and e given the large distance between the ends of
these helices, but the additional masses at the ends of helices
a–c probably do represent interhelical loops. However, contribu-
tions to all these membrane surface densities from ordered lipid
head groups, or even perhaps porphyrin-like substrates copuri-
fied with the protein, cannot be wholly excluded.
DISCUSSION
Since its discovery as the ‘‘peripheral benzodiazepine receptor’’
in 1977 (Braestrup et al., 1977; Braestrup and Squires, 1977),
TSPO has attracted considerable interest from both the
(C) Scatter plot of length and width of lipid structures present in the ice-
embedded sample. The 25 nm wide tubes constitute two thirds of the sample
and vesicles make up the remainder as shown in the pie chart (top inset). The
distribution of tube lengths ranged from 100 to 1000 nm (bottom inset).
(D) Comparison of an unprocessed cryo-EM image of a tube segment (left) with
an average of the aligned image segments of the same azimuthal views
(center) and the matched projection from the 3D image reconstruction, which
was convoluted by its corresponding CTF (right). The top and bottom row differ
in their azimuthal view by a rotation of 16� about the tube axis.
(E) Width profiles correspond to the images displayed above (left and center).
The pixel rows perpendicular to the tube axis were added to yield an averaged
width profile of the tubes, which shows that all included segments contain
tubes with a width of 25 nm (left). Averaged projection classes give rise to
a less noisy profile (center).
(F) Three-dimensional surface presentation of a TspO segment reconstructed
at 10 A resolution.
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Figure 5. Grayscale Density Slices of the
TspO 3D Reconstruction
(A) A slice of density through the helical tube with
the inset depicting the orientation of the 4.8 A thick
slices depicted in (B)–(K).
(B–G) TspO viewed parallel to the membrane
plane; slices of density were made perpendicular
to the helical axis of the tube, at various distances
from the center of the dimer (D), including �9.6 A
(B), �4.8 A (C), 4.8 A (E), 9.6 A (F), and 14.4 A (G).
(H–K) The TspO dimer viewed perpendicular to the
membrane plane; radial slices of density were
taken through the membrane plane at positions
�7.2 A (H), �2.4 A (I), +2.4 A (J), and +7.2 A (K)
with respect to the center of the bilayer, with +
being toward the external surface of the tube.
The density interpreted as one molecule of TspO
has been circled.
Structure
Cryo-EM Structure of TspO
pharmaceutical industry and academic research laboratories.
High affinity drugs for the human homolog of the TSPO/PBR
family have been developed for clinical applications and to eluci-
date its cellular functions, along with its involvement in physio-
logical processes and pathophysiological conditions (Gavish
et al., 1999; Papadopoulos et al., 2006a; Scarf et al., 2009).
Indeed, much of what we have learnt about TSPO/PBR proteins
was inferred from indirect pharmacological evidence (Gavish
et al., 1999; Papadopoulos et al., 2006a). Only a few studies
have attempted to address the structure of TSPO (Murail et al.,
2008) and most of these have addressed the structural proper-
ties of drug binding sites as inferred from structure-activity
assays (Anzini et al., 2001; Cinone et al., 2000). Consequently,
up to now most of the questions regarding how the members
of this protein family are organized on the molecular level remain
unresolved. The low-resolution reconstruction presented here
provides a first glimpse of the architecture of TspO in its native
environment, the lipid bilayer.
TspO from R. sphaeroides was purified to homogeneity in
DDM in a functional dimeric form, as suggested from the blue
native PAGE analysis (Figure 2) and the cryo-EM structure of
TspO (Figure 5). The observed dimeric form of TspO is consis-
tent with mutagenesis data, which showed that the mutation
W38C in TspO leads to the generation of covalent dimers due
to the formation of an intermolecular disulphide bond (Yeliseev
and Kaplan, 2000). Here, we show that TspO is probably a dimer
in the membrane based on the 3D structure of TspO at 10 A
resolution determined from cryo-EM images of helical tubes.
The repetitive unit in the tubular crystals is a bundle of five
a helices, which is consistent with the proposal that the TspO
monomer contains five transmembrane domains as predicted
from hydropathy analyses of the primary amino acid sequence
and confirmed by epitope mapping experiments (Joseph-Liau-
zun et al., 1998). The five a helices are packed into a ten helix
bundle related by a two-fold symmetry axis perpendicular to
the membrane. This dimer (Figure 6) is the most likely of all the
possible dimers in the crystal structure to be of physiological
relevance, due to the close proximity of the two monomers
682 Structure 18, 677–687, June 9, 2010 ª2010 Elsevier Ltd All rights
within the transmembrane region and the relatively large inter-
face between them. Surrounding this dimer are regions in the
crystal that show no strong density and are therefore predicted
to contain lipid, although strong density is present at the pre-
dicted membrane bilayer surfaces, particularly on the outside
face of the tube. The origin of this density is unclear, but it
may represent ordered regions of either the N or C termini that
mediate crystal contacts between neighboring TspO dimers.
The densities for the transmembrane a helices are sometimes
weaker than we might have anticipated, especially in the center
of the lipid bilayer, as is seen for helices a, b, and c. These
helices were modeled as continuous straight helices through
the membrane, which is possible if the discontinuities in the
density have arisen because of the low resolution of the data
or high helix mobility. Another possibility that cannot be entirely
discounted is that the helices contain regions that are non-
helical as seen in many membrane protein structures (Gouaux
and Mackinnon, 2005). Careful inspection of the density map
with adjusted contour levels reveals presence of weak connect-
ing density in those areas where discontinuities are observed,
lending support to our interpretation of the map (see Figure S4).
However, higher resolution will be required to fully understand
the structure.
Presently, we cannot assign the amino acid sequence of TspO
to particular a helices in our low-resolution model. In addition,
there is insufficient unambiguous data to suggest the order of
helix-helix connectivity in TspO, which makes building a model
of TspO from our structure more difficult; the model built of
EmrE from the 7.5 A resolution cryo-EM structure was facilitated
by the presence of clear connectivity between two of the trans-
membrane a helices (Fleishman et al., 2006). However, it is
tempting to suggest that transmembrane segment 2 of TspO
may be equivalent to the position of helix b in the reconstruction
(Figure 6). This suggestion is based on the observation that
residue W38 in the loop near the edge of the transmembrane
helix 2 of TspO forms covalent dimers when mutated to cysteine
(Yeliseev and Kaplan, 2000). Helices b and b0 in the recon-
struction make the closest intermonomer contacts and it is
reserved
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Figure 6. Interpretation of the Experimental Density Map
(A) A view perpendicular to the membrane plane of the TpsO dimer with a helices fitted into the density map by eye. Red and blue a helices represent individual
monomers of TspO and they are labeled arbitrarily a–e and a0–e0.
(B) View parallel to the membrane plane of the TspO dimer and after a 40� rotation to show the highly tilted helices e and e0.
(C) View perpendicular to the membrane plane.
(D) TspO monomer viewed parallel to the membrane plane, from the perspective of the dimer interface formed by helices a and b and a0 and b0.
(E) TspO monomer viewed from the lipid bilayer. The experimental density maps in all the panels was contoured at 1.5 s.
Structure
Cryo-EM Structure of TspO
conceivable that a crosslink between residues C38 of the two
monomers may occur in this region.
The dimeric structure of TspO raises questions regarding the
mechanism of transport of substrates across the membrane,
the stoichiometry of substrate and inhibitor binding and where
these bind in the transporter. In most structures of solute trans-
porters that contain six or more a helices, there is one transloca-
tion pathway per polypeptide chain, regardless of whether the
protein forms a monomer, trimer, or tetramer, although there
are a few exceptions, e.g., Sav1866 (Dawson and Locher,
2006). Another example of a transporter where the binding site
is formed at the interface between monomers is EmrE, which
contains four transmembrane a helices per monomer, which
combine to form an asymmetric homodimer; each dimer binds
one molecule of substrate at the interface between the two
monomers (Korkhov and Tate, 2009; Ma and Chang, 2007; Ubar-
retxena-Belandia et al., 2003; Ubarretxena-Belandia and Tate,
2004). A small molecule binding site is typically formed from
a crevice in a bundle of helices. In the structure of the TspO dimer
(Figure 6) the most likely substrate binding pocket is, therefore,
between helices d and e and between d0 and e0,which implies
that there are two binding sites per dimer. This crevice also
appears to be relatively accessible from at least one leaflet of
the lipid bilayer, which is characteristic of multidrug and lipid
transporters like EmrE (Ubarretxena-Belandia et al., 2003),
Sav1866 (Dawson and Locher, 2006), and P-glycoprotein (Aller
Structure 18
et al., 2009). It is also consistent with the proposal that the
TSPO family members can transport large hydrophobic com-
pounds such as sterols and porphyrins (Papadopoulos et al.,
2006a). The other possibility is that the binding site is at the
monomer-monomer interface, as seen in EmrE, which would
imply that one substrate molecule binds per dimer. However,
this appears to be less likely because this interface in TspO
appears relatively flat with the major interactions mediated by
helix b from each monomer. It is, therefore, clearly desirable to
determine the stoichiometry of substrate binding to either
TspO or to one of its homologs, as this would allow the distinc-
tion between these two hypotheses. Similar data were important
in defining the functional unit of EmrE as a homodimer (Butler
et al., 2004), but this required an accurate radioligand binding
assay, using a substrate that bound with high affinity, coupled
to amino acid analysis of the purified protein. The lack of high-
affinity radioligands for TspO precludes such a study in our
work, although this may be possible for mammalian TSPO, which
has a greater diversity of radiolabeled substrates and inhibitors,
provided that TSPO can be overexpressed and purified in a fully
functional form.
The low-resolution cryo-EM structure of TspO we present here
is suggestive of two binding sites per dimer, which, in turn, allows
for the possibility of cooperativity during substrate transport and
potential effects of allosteric modulators. Given that human
TSPO is 33.5% identical to R. sphaeroides TspO, it is likely
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Structure
Cryo-EM Structure of TspO
that human TSPO will have a similar architecture to bacterial
TspO, implying that human TSPO could also have two substrate
translocation pathways and thus two potential drug binding sites
in close proximity. Thus, the structure could explain the previ-
ously observed allosterism of ligand binding to TSPO/PBR
proteins (Masonis and McCarthy, 1996) and the existence of
several classes of drug binding sites (Boujrad et al., 1994,
1996). Experimental determination of the oligomeric state of
human TSPO will therefore be extremely informative. The struc-
ture of R. sphaeroides TspO at higher resolution will help to
define further the pathway of substrate translocation and will
provide a valuable framework for future studies on this family
of proteins.
EXPERIMENTAL PROCEDURES
Reagents
Chemicals were purchased from Sigma-Aldrich. Hemin, PPIX, and cPPIII were
purchased from Frontier Scientific. DDM (Anagrade) was purchased from Ana-
trace. E. coli polar lipids were purchased from Avanti Polar Lipids. TspO was
isolated by PCR from plasmid pUI2701, generously provided by S. Kaplan,
and cloned into plasmid pET29 (Novagen).
Protein Expression and Purification
TspO was cloned into the NdeI and XhoI sites restriction sites of plasmid
pET29 in E. coli strain BL21(DE3) for expression from the T7 promoter. Briefly,
cells transformed with the pET29-TspO plasmid were grown to an OD600 of
2.0–2.5 in 2xTY medium and induced with 0.5 mM IPTG for 3 hr at 32�C.
The cells were harvested by centrifugation (15 min, 5000 3 g, 4�C). The pellets
were resuspended in buffer A on ice [20 mM Tris (pH 7.5) and 200 mM NaCl]
and the cells broken open using a continuous flow cell disruptor. After a brief
spin at low speed (30 min, 10,000 3 g, 4�C), the cell membranes were
collected using a Ti45 rotor in an ultracentrifuge (2 hr, 170,000 3 g, 4�C), resus-
pended in buffer A, flash frozen in liquid nitrogen, and stored at �80�C. For
purification, the membranes were solubilized in buffer A supplemented with
2% DDM, and the insoluble material was removed by ultracentrifugation as
before. The protein was purified using a His6 tag on NiNTA-agarose (QIAGEN),
according to the manufacturer’s protocol. The protein eluted from the
Ni-affinity column was separated from the aggregates using gel filtration in
20 mM Tris (pH 7.5), 30 mM NaCl, and 0.025% DDM.
Inner and Outer Membrane Separation
To separate the inner and outer membranes of E. coli, a two-step sucrose
gradient fractionation protocol was used. The membrane preparations from
TspO-expressing cells were mixed with sucrose (20% final concentration)
and layered onto a pre-formed 70%–60% two-step sucrose gradient contain-
ing equivalent amounts of buffer and salt in a centrifuge tube (final volume ratio
for the solutions with different sucrose concentrations of 1:1:1). Following
centrifugation (16 hr, 260,000 3 g, 4�C) the bands corresponding to inner
membranes (golden layer between the 20% and 60% sucrose layers) and
outer membranes (white layer between the 60% and 70% sucrose layers)
were collected, diluted with buffer A, and centrifuged (2 hr, 310,000 3 g,
4�C). The final pellet was resuspended in buffer A and used for SDS-PAGE
and western blot analysis, according to standard procedures. For TspO-His6
detection, an HRP-conjugated His tag antibody (Novagen) was used, following
the vendor’s instructions.
Blue Native PAGE
To determine the oligomeric state of the protein in detergent micelles, blue
native PAGE was performed (Schagger and von Jagow, 1991). The 4%–
16% gradient Novex Bis-Tris gels (Invitrogen) and the commercially available
cathode and anode buffers for blue native PAGE (Invitrogen) were used.
Protein samples in DDM were mixed with the loading buffer and loaded on
a gel (5–10 mg protein/lane). Electrophoresis (performed at 4�C for 4–6 hr)
and gel destaining were performed according to the vendor’s instructions.
684 Structure 18, 677–687, June 9, 2010 ª2010 Elsevier Ltd All rights
Analytical Size Exclusion Chromatography
A purified TspO sample, concentrated to 1 mg/ml, was loaded on a Superdex
200 10/300 GL column, pre-equilibrated with buffer composed of 20 mM Tris
(pH 7.5), 200 mM NaCl, and 0.025% DDM. The UV absorbance at 280 nm was
monitored during chromatography and plotted against time (in minutes).
Binding Assays
To assess the interactions between purified TspO and porphyrins, intrinsic
tryptophan-fluorescence quenching was used. In brief, purified TspO in
DDM at a final concentration of 2.5 mM was titrated with increasing amounts
of heme in the desired concentration range. Each titration point was followed
by a spectrum measurement (excitation at 295 nm, emission at 300–400 nm)
and detection of tryptophan fluorescence. Arbitrary fluorescence units at
340 nm were plotted against ligand concentration to evaluate the fluorescence
quenching. The apparent dissociation constants, Kapp, were estimated using
a simple one-site binding model in GraphPad Prism.
Crystallization of TspO Tubes
Reconstitution of the purified TspO into helical tubular crystals was performed
in a setup similar to that used previously for EmrE (Korkhov and Tate,
2008; Tate et al., 2001). The protein preparations at final concentrations of
0.5–1 mg/ml were incubated with E. coli polar lipid extract at lipid/protein ratios
of 0.2–0.6 in Slide-A-Lyzer cassettes (Pierce) submerged in 100 ml beakers
filled with dialysis buffer [20 mM Tris (pH 7.5), 100 mM NaCl, and 2 mM
EDTA] for 3 days, with daily exchange of dialysis buffer. The tubular crystals
obtained this way were used directly for cryo-EM.
Electron Microscopy
For freezing and cryo-EM, tubes of TspO at a concentration of 0.5 mg/ml in
reconstitution buffer were pipetted on to a glow-discharged holey carbon
grid (Agar Scientific). The solution was blotted from the opposite side of the
grid with a strip of filter paper and the grid was plunged into a reservoir of liquid
ethane. Grids frozen this way were then cryo-transferred into storage boxes.
Standard cryo-transfer procedures were then used with a Gatan 626 cryo-
stage for electron microscopy. The tubes were analyzed with a Tecnai F20
electron microscope operating at an accelerating voltage of 200 kV, using
standard low-dose imaging conditions (10–15 e/A�2). Images were acquired
on film, using flood-beam illumination at a magnification of 50,000 with an
underfocus between 0.7 and 2.3 mm. For further analysis, 36 micrographs
were digitized using a home-built KZA scanner (Henderson et al., 2007) with
a 6 m step size and processed as described below. For the hand determina-
tion, TspO tubes were negatively stained using a 0.75% uranyl formate solu-
tion (Ohi et al., 2004); we recorded images of these tubes on an FEI Tecnai
T12 microscope at a magnification of 42,000 using a 2 3 2k CCD camera.
The final pixel size on the specimen of 3.25 A/pixel was calibrated by
measuring the (23 A)�1 layer line of 18 tobacco mosaic virus (TMV) reference
specimens.
Image Processing and 3D Image Reconstruction
The structure of TspO lipid tubes was determined using a recently developed
single particle-based helical reconstruction algorithm (Sachse et al., 2007).
Initially, the method was based on iterative helical real-space reconstruction
(Egelman, 2007) but has been significantly extended by applying a full correc-
tion of the contrast-transfer function (CTF) of the microscope, imposing
restraints on the alignment and a new symmetrization procedure (Sachse
et al., 2007). This improved single-particle reconstruction procedure imposes
helical symmetry in real space and thus requires precise knowledge of param-
eters of helical symmetry. Therefore, we derived initial parameters of helical
symmetry from image analysis of the helical diffraction pattern of averaged
class sums (Crowther et al., 1996) and further refined it using the single-particle
method described below.
Fourier-Bessel Analysis of Diffraction Pattern and Determination
of Helical Symmetry Parameters
First, we inspected the helical diffraction pattern on single tubes. For this anal-
ysis, TspO lipid tubules were boxed, padded, and floated using Ximdisp
(Smith, 1999) and were Fourier transformed. Selected tubes possessed three
layer lines at (102 A)�1, (48 A)�1, and (32 A)�1. Because of the limited signal
reserved
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Structure
Cryo-EM Structure of TspO
present in single tube images, we classified the segmented tube stack using
multivariate statistical analysis and further analyzed two well-ordered classes
with apparent stacking striations from a total of twelve classes (Figure S1B).
The remaining classes did not show clear stacking or moire features owing
to inherent crystal disorder and insufficient separation of different azimuthal
or out-of-plane views. The amplitudes and phases of the best two classes
could be readily analyzed and the helical lattice was indexed. The (32 A)�1 layer
line exhibits a maximum on the meridian and has, therefore, a Bessel order
of 0. The reciprocal radius of the (102 A)�1 and (48 A)�1 layer line maxima
and the presence of equal phases at the layer line peaks mirrored along the
meridian indicate a Bessel order of 12 with opposite signs, respectively
(Figure S1C). Hence, the derived lattice consists of helically related stacked
rings, each containing a 12-fold rotational symmetry (Figure S1D). This config-
uration resulted in helical symmetry parameters of 9.6� and 32.4 A. Because of
the possible ambiguity in Bessel order determination, we also tested single-
particle helical reconstructions with 10, 11, 13, and 14 start helical lattices,
but none of these reconstructions were meaningful (data not shown). In
contrast, the diffraction pattern and side views of the 12 start helical lattice
model showed maximum correlation with the diffraction of the experimental
image data and their class averages (data not shown). In addition, we also per-
formed a classical Fourier-Bessel 3D reconstruction using three selected
tubes with good diffraction (Crowther et al., 1996). The resulting 30 A resolution
map is comparable with the low-pass-filtered version of the single-particle
reconstruction (data not shown).
Refinement of Helical Symmetry Parameters
Optimization of helical symmetry parameters started at a helical rise of 32.4 A
and a helical rotation of 9.6�. The grid searches covered 25 combinations
ranging from 32.0–32.8 A in helical rise to 9.2–10.0� in helical rotation (Figures
S1F and S1G). For each symmetry combination, four cycles was sufficient to
produce a stable 3D reconstruction with each set of fixed symmetry parame-
ters. We evaluated the cross-correlation between the power spectra sum of
in-plane rotated segments and the simulated power spectrum of the 3D recon-
struction of the particular combination of helical symmetry parameters. The
optimal combination of helical symmetry parameters was determined by
a grid search subdivided into a finer grid until the cross-correlation converged.
In order to minimize the influence of noise and resolution-dependent amplitude
scaling, we correlated resolution rings individually between 100 A and 16 A and
used their average as a final measure (Figure S1G). Finally, a helical rise of
32.67 A with a rotation of 9.53� produced maximum correlation between the
sum of experimental power spectra and the simulated symmetry-imposed
power spectrum of the 3D reconstruction (Figures S1D and S1E).
Single-Particle Helical Reconstruction of TspO at 10 A Resolution
TspO tubules of a width of�250 A were segmented into overlapping images of
108 3 108 nm using Boxer from the EMAN software suite (Ludtke et al., 1999).
We used a step size of 32 A that directly corresponds to the repeat of the
stacked rings. The CTF of the electron micrographs was determined using
CTFFIND and subsequently refined by CTFTILT to yield segment-specific
CTF parameters (Mindell and Grigorieff, 2003). Further image processing
was performed using SPIDER software (Frank et al., 1996). For CTF correction,
each segment was convoluted by the corresponding CTF and then subjected
to iterative cycles of projection matching. Finally, angles around the helical axis
were sampled in 2� intervals and out-of-plane tilt of the tubules was taken into
account ±12� at 2� increments. Using the determined alignment parameters,
each image segment was inserted 12 times into the 3D image reconstruction
corresponding to the 12-fold rotational symmetry present in the ring. Asym-
metric units related by the helical symmetry were already included as separate
images on the stack due to the 32 A step size used during the segmentation
procedure. In addition to the helical lattice, we exploited the two-fold
symmetry present within the TspO dimer. During projection matching, we
found that there was no preferred polarity among the segments of a single
tubule. Therefore, we aligned each segment twice with an in-plane rotation
differing by 180� and included them in the 3D reconstruction. Finally, the
volume was corrected by division of the average 3D value of CTF2 in addition
to a Wiener filter constant of 1% of the maximum amplitude. In order to mini-
mize noise, helical symmetry was also imposed on the final 3D volume after
each cycle. We excluded segments whose (32 A)�1 ring correlation of its power
Structure 18
spectrum with the sum of all in-plane rotated power spectra was below 0.45.
Because images were taken at low underfocus between 0.7 and 2.3 mm, the
ring correlation at 32 A is only minimally affected by CTF variation. Excluded
segments with low ring cross-correlation showed weak layer-line diffraction.
We included only 35%, or 2610, of the 7542 segments in the final 3D image
reconstruction of 72 3 72 3 72 nm in size. The resolution of the final map
was estimated using Fourier shell correlation criteria at 0.5/0.143 cut-offs
yielding 10.2/7.8 A, respectively (Figure S3). It should be noted that the numer-
ical resolution cutoff appeared sensitive to the tightness of the applied mask. In
order to avoid overestimation of the resolution, we dilated the structural mask
by an additional 10 A. The resolution estimate of 10.2 A corresponds to the
detail expected from a 10 A resolution map. More image processing statistics
are displayed in Table 1. The final volume was corrected for amplitude decay
with a B factor of �400 A2 while applying a figure-of-merit weighting scheme
(Rosenthal and Henderson, 2003).
Hand Determination
We determined the hand of TspO tubes using one-sided negative staining and
helical diffraction analysis. Tubes from negatively stained grids showed signif-
icant asymmetry in their layer lines between adjacent quadrants in their power
spectrum due to variation of stain thickness across the grid. Frequently, stain
covers only the side of the particle facing the carbon (Klug, 1965; Klug and
Finch, 1965). In the case of TMV, an asymmetric layer line was observed at
69 A. According to the TMV reference structure (Finch, 1972), the (69 A)�1 layer
line corresponds to a Bessel order of 16 with left-handed helicity (Figure S2B).
As expected, when imaging the specimen with the carbon side facing the
electron gun, the striations present on the near side give rise to a stronger layer
line on the right-hand side of the upper quadrant of the power spectrum
(Figure S2C). In order to exclude asymmetric effects arising from different
azimuthal views, we performed a control simulation, which shows that the
(69 A)�1 layer line is not subject to intensity variation during rotation around
the helical axis or around the out-of-plane tilt axis (data not shown). The anal-
ysis of one-sided TspO tubules yields a (102 A)�1 layer line, which shows
a stronger reflection on the right-hand side of the upper quadrant, thus it
also possesses left-handed helicity (Figures S2E and S2F). Therefore the
(48 A)�1 layer line carries the opposite sign according to the helical lattice.
This asymmetry in the layer line is less obvious than for the (102 A)�1 layer
line. In order to increase the signal of the layer lines and compensate for
long-range bending, overlapping segments of the tubes were excised using
BOXER (Ludtke et al., 1999), rotated in the image plane, and Fourier trans-
formed, and their power spectra were averaged. By analogy, we performed
these tests for the structure of TspO and came to the same conclusion that
the observed asymmetry in the (102 A)�1 layer line is not due to different
azimuthal or out-of-plane views. We also performed rotary shadowing analysis
and tilting experiments to try and determine the hand of the structure indepen-
dently; results for both methods were inconclusive. First, no lattice features
were revealed by the smooth-surfaced helix in the platinum shadow. Second,
we tested tilting the helix perpendicular to the helical axis (Finch, 1972), but,
due to the relatively long helical pitch, insufficient asymmetry was shown by
either the left or right side of the projections.
ACCESSION NUMBERS
The reconstructed TspO map has been deposited at the EMDB databank with
accession number EMD-1698.
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures and can be found with this
article online at doi:10.1016/j.str.2010.03.001.
ACKNOWLEDGMENTS
We would like to thank both R. Henderson and N. Unwin for helpful comments
on image processing and manuscript preparation. The work was supported
by core funding from the Medical Research Council and by an EC FP7 grant
for the EDICT consortium (HEALTH-201924). V.M.K. was supported by
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Structure
Cryo-EM Structure of TspO
a Federation of European Biochemical Societies Long-Term Fellowship and
C.S. was the recipient of a European Molecular Biology Organization Long-
Term Fellowship. The authors declare that they have no conflict of interest.
Received: October 15, 2009
Revised: February 8, 2010
Accepted: March 2, 2010
Published: June 8, 2010
REFERENCES
Aller, S.G., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R., Harrell, P.M.,
Trinh, Y.T., Zhang, Q., Urbatsch, I.L., and Chang, G. (2009). Structure of
P-glycoprotein reveals a molecular basis for poly-specific drug binding.
Science 323, 1718–1722.
Anzini, M., Cappelli, A., Vomero, S., Seeber, M., Menziani, M.C., Langer, T.,
Hagen, B., Manzoni, C., and Bourguignon, J.J. (2001). Mapping and fitting
the peripheral benzodiazepine receptor binding site by carboxamide deriva-
tives. Comparison of different approaches to quantitative ligand-receptor
interaction modeling. J. Med. Chem. 44, 1134–1150.
Bogan, R.L., Davis, T.L., and Niswender, G.D. (2007). Peripheral-type benzo-
diazepine receptor (PBR) aggregation and absence of steroidogenic acute
regulatory protein (StAR)/PBR association in the mitochondrial membrane as
determined by bioluminescence resonance energy transfer (BRET). J. Steroid
Biochem. Mol. Biol. 104, 61–67.
Boujrad, N., Gaillard, J.L., Garnier, M., and Papadopoulos, V. (1994). Acute
action of choriogonadotropin on Leydig tumor cells: induction of a higher
affinity benzodiazepine-binding site related to steroid biosynthesis. Endocri-
nology 135, 1576–1583.
Boujrad, N., Vidic, B., and Papadopoulos, V. (1996). Acute action of
choriogonadotropin on Leydig tumor cells: changes in the topography of the
mitochondrial peripheral-type benzodiazepine receptor. Endocrinology 137,
5727–5730.
Braestrup, C., and Squires, R.F. (1977). Specific benzodiazepine receptors in
rat brain characterized by high-affinity (3H)diazepam binding. Proc. Natl. Acad.
Sci. USA 74, 3805–3809.
Braestrup, C., Albrechtsen, R., and Squires, R.F. (1977). High densities of
benzodiazepine receptors in human cortical areas. Nature 269, 702–704.
Butler, P.J., Ubarretxena-Belandia, I., Warne, T., and Tate, C.G. (2004). The
Escherichia coli multidrug transporter EmrE is a dimer in the detergent-
solubilised state. J. Mol. Biol. 340, 797–808.
Cinone, N., Hotje, H.D., and Carotti, A. (2000). Development of a unique 3D
interaction model of endogenous and synthetic peripheral benzodiazepine
receptor ligands. J. Comput. Aided Mol. Des. 14, 753–768.
Crowther, R.A., Henderson, R., and Smith, J.M. (1996). MRC image process-
ing programs. J. Struct. Biol. 116, 9–16.
Dawson, R.J., and Locher, K.P. (2006). Structure of a bacterial multidrug ABC
transporter. Nature 443, 180–185.
Delavoie, F., Li, H., Hardwick, M., Robert, J.C., Giatzakis, C., Peranzi, G., Yao,
Z.X., Maccario, J., Lacapere, J.J., and Papadopoulos, V. (2003). In vivo
and in vitro peripheral-type benzodiazepine receptor polymerization: func-
tional significance in drug ligand and cholesterol binding. Biochemistry 42,
4506–4519.
Dong, C., Beis, K., Nesper, J., Brunkan-Lamontagne, A.L., Clarke, B.R.,
Whitfield, C., and Naismith, J.H. (2006). Wza the translocon for E. coli capsular
polysaccharides defines a new class of membrane protein. Nature 444,
226–229.
Egelman, E.H. (2007). The iterative helical real space reconstruction method:
surmounting the problems posed by real polymers. J. Struct. Biol. 157, 83–94.
Finch, J.T. (1972). The hand of the helix of tobacco virus. J. Mol. Biol. 66,
291–294.
Fleishman, S.J., Harrington, S.E., Enosh, A., Halperin, D., Tate, C.G., and
Ben-Tal, N. (2006). Quasi-symmetry in the cryo-EM structure of EmrE provides
the key to modeling its transmembrane domain. J. Mol. Biol. 364, 54–67.
686 Structure 18, 677–687, June 9, 2010 ª2010 Elsevier Ltd All rights
Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M., and Leith,
A. (1996). SPIDER and WEB: processing and visualization of images in 3D
electron microscopy and related fields. J. Struct. Biol. 116, 190–199.
Galiegue, S., Casellas, P., Kramar, A., Tinel, N., and Simony-Lafontaine, J.
(2004). Immunohistochemical assessment of the peripheral benzodiazepine
receptor in breast cancer and its relationship with survival. Clin. Cancer Res.
10, 2058–2064.
Garnier, M., Dimchev, A.B., Boujrad, N., Price, J.M., Musto, N.A., and
Papadopoulos, V. (1994). In vitro reconstitution of a functional peripheral-
type benzodiazepine receptor from mouse Leydig tumor cells. Mol. Pharma-
col. 45, 201–211.
Gavish, M., Bachman, I., Shoukrun, R., Katz, Y., Veenman, L., Weisinger, G.,
and Weizman, A. (1999). Enigma of the peripheral benzodiazepine receptor.
Pharmacol. Rev. 51, 629–650.
Gouaux, E., and Mackinnon, R. (2005). Principles of selective ion transport in
channels and pumps. Science 310, 1461–1465.
Han, Z., Slack, R.S., Li, W., and Papadopoulos, V. (2003). Expression of
peripheral benzodiazepine receptor (PBR) in human tumors: relationship to
breast, colorectal, and prostate tumor progression. J. Recept. Signal Trans-
duct. Res. 23, 225–238.
Henderson, R., Cattermole, D., McMullan, G., Scotcher, S., Fordham, M.,
Amos, W.B., and Faruqi, A.R. (2007). Digitisation of electron microscope films:
six useful tests applied to three film scanners. Ultramicroscopy 107, 73–80.
Joseph-Liauzun, E., Delmas, P., Shire, D., and Ferrara, P. (1998). Topological
analysis of the peripheral benzodiazepine receptor in yeast mitochondrial
membranes supports a five-transmembrane structure. J. Biol. Chem. 273,
2146–2152.
Klug, A. (1965). Structure of viruses of the papilloma-polyoma type. II.
Comments on other work. J. Mol. Biol. 11, 424–431.
Klug, A., and Finch, J.T. (1965). Structure of viruses of the papilloma-polyoma
type. I. Human wart virus. J. Mol. Biol. 11, 403–423.
Korkhov, V.M., and Tate, C.G. (2008). Electron crystallography reveals plas-
ticity within the drug binding site of the small multidrug transporter EmrE.
J. Mol. Biol. 377, 1094–1103.
Korkhov, V.M., and Tate, C.G. (2009). An emerging consensus for the structure
of EmrE. Acta Crystallogr. D Biol. Crystallogr. 65, 186–192.
Lacapere, J.J., Delavoie, F., Li, H., Peranzi, G., Maccario, J., Papadopoulos,
V., and Vidic, B. (2001). Structural and functional study of reconstituted
peripheral benzodiazepine receptor. Biochem. Biophys. Res. Commun. 284,
536–541.
Lacapere, J.J., and Papadopoulos, V. (2003). Peripheral-type benzodiazepine
receptor: structure and function of a cholesterol-binding protein in steroid and
bile acid biosynthesis. Steroids 68, 569–585.
Li, H., and Papadopoulos, V. (1998). Peripheral-type benzodiazepine receptor
function in cholesterol transport. Identification of a putative cholesterol recog-
nition/interaction amino acid sequence and consensus pattern. Endocrinology
139, 4991–4997.
Li, H., Yao, Z., Degenhardt, B., Teper, G., and Papadopoulos, V. (2001).
Cholesterol binding at the cholesterol recognition/interaction amino acid
consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibi-
tion of steroidogenesis by an HIV TAT-CRAC peptide. Proc. Natl. Acad. Sci.
USA 98, 1267–1272.
Low, H.H., Sachse, C., Amos, L.A., and Lowe, J. (2009). Structure of a bacterial
dynamin-like protein lipid tube provides a mechanism for assembly and
membrane curving. Cell 139, 1342–1352.
Ludtke, S.J., Baldwin, P.R., and Chiu, W. (1999). EMAN: semiautomated soft-
ware for high-resolution single-particle reconstructions. J. Struct. Biol. 128,
82–97.
Ma, C., and Chang, G. (2007). Structure of the multidrug resistance efflux
transporter EmrE from Escherichia coli. Proc. Natl. Acad. Sci. USA 104, 3668.
Maaser, K., Grabowski, P., Sutter, A.P., Hopfner, M., Foss, H.D., Stein, H.,
Berger, G., Gavish, M., Zeitz, M., and Scherubl, H. (2002). Overexpression of
the peripheral benzodiazepine receptor is a relevant prognostic factor in stage
III colorectal cancer. Clin. Cancer Res. 8, 3205–3209.
reserved
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Structure
Cryo-EM Structure of TspO
Maaser, K., Hopfner, M., Jansen, A., Weisinger, G., Gavish, M., Kozikowski,
A.P., Weizman, A., Carayon, P., Riecken, E.O., Zeitz, M., and Scherubl, H.
(2001). Specific ligands of the peripheral benzodiazepine receptor induce
apoptosis and cell cycle arrest in human colorectal cancer cells. Br. J. Cancer
85, 1771–1780.
Masonis, A.E., and McCarthy, M.P. (1996). Direct interactions of androgenic/
anabolic steroids with the peripheral benzodiazepine receptor in rat brain:
implications for the psychological and physiological manifestations of andro-
genic/anabolic steroid abuse. J. Steroid Biochem. Mol. Biol. 58, 551–555.
Mindell, J.A., and Grigorieff, N. (2003). Accurate determination of local defocus
and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347.
Murail, S., Robert, J.C., Coic, Y.M., Neumann, J.M., Ostuni, M.A., Yao, Z.X.,
Papadopoulos, V., Jamin, N., and Lacapere, J.J. (2008). Secondary and
tertiary structures of the transmembrane domains of the translocator protein
TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand
binding. Biochim. Biophys. Acta 1778, 1375–1381.
O’Hara, M.F., Nibbio, B.J., Craig, R.C., Nemeth, K.R., Charlap, J.H., and
Knudsen, T.B. (2003). Mitochondrial benzodiazepine receptors regulate
oxygen homeostasis in the early mouse embryo. Reprod. Toxicol. 17, 365–375.
Ohi, M., Li, Y., Cheng, Y., and Walz, T. (2004). Negative staining and image
classification - powerful tools in modern electron microscopy. Biol. Proced.
Online 6, 23–34.
Papadopoulos, V., Boujrad, N., Ikonomovic, M.D., Ferrara, P., and Vidic, B.
(1994). Topography of the Leydig cell mitochondrial peripheral-type benzodi-
azepine receptor. Mol. Cell. Endocrinol. 104, R5–R9.
Papadopoulos, V., Baraldi, M., Guilarte, T.R., Knudsen, T.B., Lacapere, J.J.,
Lindemann, P., Norenberg, M.D., Nutt, D., Weizman, A., Zhang, M.R., and
Gavish, M. (2006a). Translocator protein (18kDa): new nomenclature for the
peripheral-type benzodiazepine receptor based on its structure and molecular
function. Trends Pharmacol. Sci. 27, 402–409.
Papadopoulos, V., Lecanu, L., Brown, R.C., Han, Z., and Yao, Z.X. (2006b).
Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis, neuro-
pathology and neurological disorders. Neuroscience 138, 749–756.
Rone, M.B., Liu, J., Blonder, J., Ye, X., Veenstra, T.D., Young, J.C., and
Papadopoulos, V. (2009). Targeting and insertion of the cholesterol-binding
translocator protein into the outer mitochondrial membrane. Biochemistry
48, 6909–6920.
Rosenthal, P.B., and Henderson, R. (2003). Optimal determination of particle
orientation, absolute hand, and contrast loss in single-particle electron cryomi-
croscopy. J. Mol. Biol. 333, 721–745.
Sachse, C., Chen, J.Z., Coureux, P.D., Stroupe, M.E., Fandrich, M., and
Grigorieff, N. (2007). High-resolution electron microscopy of helical speci-
mens: a fresh look at tobacco mosaic virus. J. Mol. Biol. 371, 812–835.
Scarf, A.M., Ittner, L.M., and Kassiou, M. (2009). The translocator protein
(18 kDa): central nervous system disease and drug design. J. Med. Chem.
52, 581–592.
Schagger, H., and von Jagow, G. (1991). Blue native electrophoresis for
isolation of membrane protein complexes in enzymatically active form. Anal.
Biochem. 199, 223–231.
Structure 18
Smith, J.M. (1999). Ximdisp—A visualization tool to aid structure determination
from electron microscope images. J. Struct. Biol. 125, 223–228.
Taketani, S., Kohno, H., Okuda, M., Furukawa, T., and Tokunaga, R. (1994).
Induction of peripheral-type benzodiazepine receptors during differentiation
of mouse erythroleukemia cells. A possible involvement of these receptors in
heme biosynthesis. J. Biol. Chem. 269, 7527–7531.
Tate, C.G., Kunji, E.R., Lebendiker, M., and Schuldiner, S. (2001). The projec-
tion structure of EmrE, a proton-linked multidrug transporter from Escherichia
coli, at 7 A resolution. EMBO J. 20, 77–81.
Ubarretxena-Belandia, I., and Tate, C.G. (2004). New insights into the structure
and oligomeric state of the bacterial multidrug transporter EmrE: an unusual
asymmetric homo-dimer. FEBS Lett. 564, 234–238.
Ubarretxena-Belandia, I., Baldwin, J.M., Schuldiner, S., and Tate, C.G. (2003).
Three-dimensional structure of the bacterial multidrug transporter EmrE
shows it is an asymmetric homodimer. EMBO J. 22, 6175–6181.
Veenman, L., Leschiner, S., Spanier, I., Weisinger, G., Weizman, A., and
Gavish, M. (2002). PK 11195 attenuates kainic acid-induced seizures and
alterations in peripheral-type benzodiazepine receptor (PBR) protein compo-
nents in the rat brain. J. Neurochem. 80, 917–927.
Weisinger, G., Kelly-Hershkovitz, E., Veenman, L., Spanier, I., Leschiner, S.,
and Gavish, M. (2004). Peripheral benzodiazepine receptor antisense
knockout increases tumorigenicity of MA-10 Leydig cells in vivo and in vitro.
Biochemistry 43, 12315–12321.
Wendler, G., Lindemann, P., Lacapere, J.J., and Papadopoulos, V. (2003).
Protoporphyrin IX binding and transport by recombinant mouse PBR.
Biochem. Biophys. Res. Commun. 311, 847–852.
Yeliseev, A.A., and Kaplan, S. (1995). A sensory transducer homologous to the
mammalian peripheral-type benzodiazepine receptor regulates photosyn-
thetic membrane complex formation in Rhodobacter sphaeroides 2.4.1.
J. Biol. Chem. 270, 21167–21175.
Yeliseev, A.A., and Kaplan, S. (1999). A novel mechanism for the regulation of
photosynthesis gene expression by the TspO outer membrane protein of
Rhodobacter sphaeroides 2.4.1. J. Biol. Chem. 274, 21234–21243.
Yeliseev, A.A., and Kaplan, S. (2000). TspO of rhodobacter sphaeroides.
A structural and functional model for the mammalian peripheral benzodiaze-
pine receptor. J. Biol. Chem. 275, 5657–5667.
Yeliseev, A.A., Krueger, K.E., and Kaplan, S. (1997). A mammalian mitochon-
drial drug receptor functions as a bacterial ‘‘oxygen’’ sensor. Proc. Natl. Acad.
Sci. USA 94, 5101–5106.
Zeng, X., and Kaplan, S. (2001). TspO as a modulator of the repressor/antire-
pressor (PpsR/AppA) regulatory system in Rhodobacter sphaeroides 2.4.1.
J. Bacteriol. 183, 6355–6364.
Zhang, M.R., Maeda, J., Ogawa, M., Noguchi, J., Ito, T., Yoshida, Y., Okauchi,
T., Obayashi, S., Suhara, T., and Suzuki, K. (2004). Development of a new
radioligand, N-(5-fluoro-2-phenoxyphenyl)-N-(2-[18F]fluoroethyl-5-methoxy-
benzyl)acetamide, for pet imaging of peripheral benzodiazepine receptor in
primate brain. J. Med. Chem. 47, 2228–2235.
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