REFERENCES AND NOTES
1. M. Falkenberg, N. G. Larsson, C. M. Gustafsson, Annu. Rev.
Biochem. 76, 679–699 (2007).
2. N. D. Bonawitz, D. A. Clayton, G. S. Shadel, Mol. Cell 24,
3. B. M. Hällberg, N. G. Larsson, Cell Metab. 20, 226–240
4. Y. I. Morozov et al., Nucleic Acids Res. 42, 3884–3893 (2014).
5. P. H. Wanrooij et al., Nucleic Acids Res. 40, 10334–10344
6. P. H. Wanrooij, J. P. Uhler, T. Simonsson, M. Falkenberg,
C. M. Gustafsson, Proc. Natl. Acad. Sci. U.S.A. 107,
7. D. D. Chang, D. A. Clayton, Proc. Natl. Acad. Sci. U.S.A. 82,
8. D. Kang, K. Miyako, Y. Kai, T. Irie, K. Takeshige, J. Biol. Chem.
272, 15275–15279 (1997).
9. D. A. Clayton, Cell 28, 693–705 (1982).
10. J. M. Fusté et al., Mol. Cell 37, 67–78 (2010).
11. R. T. Pomerantz, M. O’Donnell, Cell Cycle 9, 2537–2543
12. M. Minczuk et al., Nucleic Acids Res. 39, 4284–4299
13. V. Epshtein, C. J. Cardinale, A. E. Ruckenstein, S. Borukhov,
E. Nudler, Mol. Cell 28, 991–1001 (2007).
14. R. M. Andrews et al., Nat. Genet. 23, 147 (1999).
15. K. Schwinghammer et al., Nat. Struct. Mol. Biol. 20, 1298–1303
16. T. H. Tahirov et al., Nature 420, 43–50 (2002).
17. I. Gusarov, E. Nudler, Cell 107, 437–449 (2001).
18. S. Shankar, A. Hatoum, J. W. Roberts, Mol. Cell 27, 914–927
19. A. Rantanen, N. G. Larsson, Hum. Reprod. 15 (suppl. 2), 86–91
20. J. St John, Biochim. Biophys. Acta 1840, 1345–1354 (2014).
21. T. Wai et al., Biol. Reprod. 83, 52–62 (2010).
22. P. J. Carling, L. M. Cree, P. F. Chinnery, Mitochondrion 11,
23. J. Van Blerkom, Mitochondrion 11, 797–813 (2011).
24. S. Monnot et al., Hum. Mol. Genet. 22, 1867–1872 (2013).
25. D. C. Wallace, Annu. Rev. Genet. 39, 359–407 (2005).
26. M. I. Ekstrand et al., Hum. Mol. Genet. 13, 935–944 (2004).
We thank K. Schwinghammer for help in cloning and isolation
of TEFM and W. McAllister and M. Gottesman for the critical
discussion and reading of the manuscript. This work was
supported in part by NIH R01GM104231.
Materials and Methods
Figs. S1 to S5
16 October 2014; accepted 23 December 2014
Structure and activity of
tryptophan-rich TSPO proteins
Youzhong Guo,1 Ravi C. Kalathur,2 Qun Liu,2,3 Brian Kloss,2 Renato Bruni,2
Christopher Ginter,2 Edda Kloppmann,2,4 Burkhard Rost,2,4 Wayne A. Hendrickson1,2,3,5*
Translocator proteins (TSPOs) bind steroids and porphyrins, and they are implicated in
many human diseases, for which they serve as biomarkers and therapeutic targets. TSPOs
have tryptophan-rich sequences that are highly conserved from bacteria to mammals. Here
we report crystal structures for Bacillus cereus TSPO (Bc TSPO) down to 1.7 Å resolution,
including a complex with the benzodiazepine-like inhibitor PK11195. We also describe
BcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation to
a previously undescribed heme derivative. We used structure-inspired mutations to
investigate reaction mechanisms, and we showed that TSPOs from Xenopus and man have
similar PpIX-directed activities. Although TSPOs have been regarded as transporters,
the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in
protection against oxidative stress.
The translocator protein (TSPO) is named for its putative roles in the transport of cholesterol, proteins, and porphyrins into mitochondria and elsewhere(1). TSPO was first identified as a peripheral-type benzodiazepine receptor in mammals (2)—attracting
attention because of popular anxiolytic benzodiazepines such as diazepam (Valium)—and the
tryptophan-rich sensory protein TspO of photo-synthetic bacteria proved to be homologous (3).
The sequences of TSPO proteins from diverse or-
ganisms are highly conserved in sequence (fig. S1)
and also in function; notably, rat TSPO can sub-
stitute for the bacterial protein as a negative
regulator of photosynthesis gene expression in
Rhodobacter sphaeroides (4).
Benzodiazepine-like chemicals, such as PK11195,
were found to bind to TSPO and regulate steroid
biosynthesis (5), and porphyrins were identified
as endogenous ligands of rat TSPO, with highest
affinity for protoporphyrin IX (PpIX) (6). Many
following studies have pursued TSPO interactions
with cholesterol and PpIX (7, 8). Most recently,
the seeming essentiality of TSPO for steroido-
genesis (9) has been challenged by the finding
that TSPO1−/− mice are viable and without de-
fects in steroid hormone biosynthesis (10), and
the presumed role in PpIX transport (11) has been
amended by the discovery that bacterial TSPOs
catalyze a photooxidative degradation of PpIX (12).
Although the underlying biochemical mecha-
nisms for its activities are not fully understood,
TSPO has elicited considerable interest for med-
icine. Roles for TSPO have been imputed in
apoptosis (13), inflammation (14), HIV biosynthesis
(15), cancer (16), Alzheimer’s disease (17), and
cardiovascular diseases (18). TSPO is a therapeutic
target (13, 18–20) and has proven useful as a pos-
itron emission tomography–imaging biomarker
(14, 17, 21, 22).
The translocator protein has been character-
ized in monomer, dimer, and higher oligomeric
states (20). An electron microscopy structure for
TSPO from R. sphaeroides (Rs TSPO) revealed a
dimer (23), and a nuclear magnetic resonance
structure for a mouse TSPO showed a monomer
comprising five transmembrane helices (24).
We produced recombinant TSPO proteins from
several bacterial and vertebrate organisms and
found Bacillus cereus TSPO (Bc TSPO) suitable
for biochemical and structural characterization
(see supplementary materials and methods). By
size-exclusion chromatography, detergent-extracted
Bc TSPO exists in at least three different oligomeric
states (fig. S2A). Apo Bc TSPO crystallized from the
monomer-dimer fraction (fig. S2, C and D) in two
different lipidic cubic phase (LCP) conditions and
also from the high-oligomer fraction as a detergent micelle; the Bc TSPO-PK11195 complex crystallized as a dimer in LCP. A structure obtained
by single-wavelength anomalous diffraction analysis from iodinated Bc TSPO (fig. S2E) was the
starting point for native structures that followed
(tables S1 and S2). Refinements yielded structures
at 1.7 Å (Fig. 1, A to E, and fig. S3) and 2.0 Å resolution for apo monomers in LCP, at 4.1 Å resolution for the apo dimer in detergent (Fig. 1F),
and at 3.5 Å resolution for the dimeric ligand
complex in LCP (Fig. 1G).
Each Bc TSPO protomer has five transmembrane (TM) a helices organized in clockwise order
(TM1-TM2-TM5-TM4-TM3), as viewed from the
C-terminal end (Fig. 1, A and B, and fig. S1) where
a short extra-membranous helix (a1,2) between
TM1 and TM2 caps a C-terminal pocket (Fig. 1C).
The electrostatic potential surface defines a
transmembrane orientation (Fig. 1C) having its
N-terminal end heavily positive, likely cytoplasmic
in B. cereus, and its C-terminal end largely negative (Fig. 1D and fig. S4, A to D). A mapping of
sequence conservation onto the molecular surface
shows a conservative band between TM2 and
TM5, a highly conserved pocket opening between
TM1 and TM2, and features more conserved at
the C-terminal end than on most of the exterior
(Fig. 1E and fig. S4, E to H).
1Department of Biochemistry and Molecular Biophysics,
Columbia University, New York, NY 10032, USA. 2The New
York Consortium on Membrane Protein Structure
(NYCOMPS), New York Structural Biology Center, 89
Convent Avenue, New York, NY 10027, USA. 3New York
Structural Biology Center, Synchrotron Beamlines,
Brookhaven National Laboratory, Upton, NY 11973, USA.
4Department of Informatics, Bioinformatics and
Computational Biology, Technische Universität München,
Garching 85748, Germany. 5Department of Physiology and
Cellular Biophysics, Columbia University, New York, NY
*Corresponding author. E-mail: firstname.lastname@example.org