Three-Dimensional Structure of TspO by Electron Cryomicroscopy of Helical Crystals

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

, 677–687, June 9, 2010 ª2010 Elsevier Ltd All rights reserved 677

mailto:[email protected]

mailto:[email protected]

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

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


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


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


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

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


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.


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


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


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

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


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


Structure


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.


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

Structure


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


http://dx.doi.org/doi:10.1016/j.str.2010.03.001

Structure


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.


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

Structure


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.


Three-Dimensional Structure of TspO by Electron Cryomicroscopy of Helical Crystals

Documents

Transcript of Three-Dimensional Structure of TspO by Electron Cryomicroscopy of Helical Crystals