Supplementary Materials 2 - Science | AAAS Materials for Structure and activity of tryptophan-rich...
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Supplementary Materials for
Structure and activity of tryptophan-rich TSPO proteins
Youzhong Guo, Ravi C. Kalathur, Qun Liu, Brian Kloss, Renato Bruni, Christopher
Ginter, Edda Kloppmann, Burkhard Rost, Wayne A. Hendrickson*
*Corresponding author. E-mail: [email protected]
Published 30 January 2015, Science 347, 551 (2015)
DOI: 10.1126/science.aaa1534
This PDF file includes:
Materials and Methods
Figs. S1 to S10
Tables S1 and S2
Full Reference List
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Materials and Methods
Target selection
We screened several homologs of human TSPO1, a target of the New York Consortium
on Membrane Protein Structure (NYCOMPS) (35), and selected the TSPO protein from Bacillus
cereus (DSM 31) GI:30021246. (BcTSPO) for analysis.
Expression and purification of BcTSPO
The gene for BcTSPO was cloned into a pMCSG7-10xHis expression vector, which
produced a 10xHis tag at the N-terminus of BcTSPO. The plasmid was transformed into
Escherichia coli strain Bl21(DE3) PlysS. Small-scale expression and purification was conducted
following the protocol developed by Bruni and Kloss (36).
For large scale expression and purification, we typically inoculated 25mL of Terrific broth
(TB) media containing 50 µg/mL ampicillin and 25 µg/mL chloramphenicol with a glycerol stock
of the BcTSPO-expressing bacteria and incubated at 37°C shaking at 250 rpm for overnight. A
2mL aliquot of the incubated culture was added into each of ten 2L Erlenmeyer flasks containing
750mL of the antibiotic-containing TB media. The 2L Erlenmeyer flasks were incubated at 37°C
shaking at 310 rpm for 3 h. Then sterile IPTG solution was added to each flask to the final
concentration of 1mM and temperature was switched to 20°C. Then, the culture was incubated
for 18 h after the switch of temperature. E. coli cell pellets were frozen at -80°C or broken
immediately with EmulsiFlex-C3 in buffer A.
Cell membranes containing overexpressed BcTSPO were prepared by two steps of
centrifugation. First, the lysed cell solution was centrifuged for 30 min at 15000g, then the
supernatant was centrifuged for 1 hour at maximum 250,000g or 45,000rpm for the type 50.2Ti
rotor from Beckman. The membrane pellets were frozen or immediately homogenized in buffer
A and solubilized by adding 10% N-dodecyl-β-D-maltopyranoside (DDM, Anatrace, Inc) stock
solution to a final concentration 2% then kept shaking gently for 1 h at 4°C. Solubilized
membrane solutions were centrifuged at maximum 150,000g, 35,000 rpm with 50.2Ti rotor from
Beckman. The supernatant was applied immediately to a 5mL pre-packed His-TrapTM HP
column (GE healthcare Life Sciences) nickel-affinity column pre-equilibrated with buffer A
(buffers are described below). The nickel-affinity column was then washed with buffer B and
buffer C until appearance of a stable UV-280 absorbance baseline. BcTSPO elution buffer D
was applied to the washed His-TrapTM HP column. Fractions were monitored continuously for
UV-280 absorbance, collected and concentrated to 0.5mL with Amicon Ultra-15 centrifugal filter
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units from Millipore. Concentrated BcTSPO solutions were briefly centrifuged before being
applied to a Superdex 200® 10/300 GL column for gel filtration with buffer E. Fractions
containing BcTSPO were collected and concentrated to around 10mg/mL for immediate
crystallization or frozen at -80°C for later use.
Buffers were prepared with compositions as follows: Buffer A: 50mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), pH7.8, 300mM NaCl, 5% glycerol, 20mM imidazole, 1mM MgCl2, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP).
Buffer B: 50mM Hepes, pH7.8, 300mM NaCl, 5% glycerol, 40mM imidazole, 5mM MgCl2, 0.1 mM TCEP, 0.05% DDM.
Buffer C: 25mM Hepes, pH7.8, 500mM NaCl, 5% glycerol, 75mM imidazole, 0.1 mM TCEP, 0.05% DDM.
Buffer D: 25mM Hepes, pH7.8, 200mM NaCl, 5% glycerol, 250mM imidazole, 0.1 mM TCEP , 0.05% DDM.
Buffer E: 40mM Hepes, pH7.8, 100mM NaCl, 0.1 mM TCEP), 0.05% DDM.
Expression and purification of eukaryotic TSPOs
Xenopus TSPO1 and human TSPO1 and TSPO2 were cloned into modified pFastBac
vectors with C-terminal 10x-His and Flag tags using ligation-independent cloning. The resulting
baculovirus transfer vectors were used to generate recombinant bacmids using the Bac-to-Bac
system (Invitrogen) and virus was rescued by transfecting purified bacmid DNA into Sf9 cells
using Cellfectin II (Invitrogen). TSPO proteins were produced by infecting suspension cultures of
Hi5 cells (Expression systems) with recombinant baculovirus at a multiplicity of infection (MOI)
of 3–5 and incubating at 27 °C, shaking at 120 r.p.m. After 72 h, the cells were collected by
centrifugation at 500g for 10 mins and the cell pellets were stored at −80 °C. The eukaryotic
TSPO was purified using the same protocol as used for purification of BcTSPO.
Crystallization
Crystallization with detergent. BcTSPO protein was screened initially for crystallization
using a Mosquito® HTS (TTP labtech) robot with commercially available crystallization kits from
Hampton Research, Emerald Biosystems and Molecular Dimension. The high oligomeric state
BcTSPO could be crystallized in several different crystallization conditions at 20 °C, but all of
these crystals diffracted poorly, most to less than 10 Å spacings. One set of conditions did prove
successful ultimately. A sitting drop containing 2µL of BcTSPO (10mg/mL) was mixed with 2µL
of solution A (0.15 M sodium formate, 0.1 M HEPES pH7.2, 18 % w/v PEG 3350) and placed
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over a well containing 80 µL of 0.1 M HEPES pH7.2, 18 % w/v PEG 3350 and 20 µL of solution
A. Crystals that appeared after approximately one week diffracted at best to 6 Å spacings;
however, after the crystal tray was left at 4 °C for approximately six months, we fortunately
found a crystal that diffracted to 4 Å spacings and survived through a full data set collection at
BNL NSLA X4C (Data set: Apo dimer). The monomer-dimer BcTSPO fraction was crystallized
with detergents DDM or N-octyl-β-D-glucopyranoside (β-OG), but these crystals all diffracted
poorly.
Lipidic cubic phase (LCP) crystallization. For crystallization by the LCP method (37, 38),
10mg/mL BcTSPO protein solution was mixed with monoolein (9.9 MAG from Nu-Check Prep.
Inc.) in a ratio of 2:3 (v/v) using a homemade LCP mixer. The mixed LCP sample was used for
crystallization screening with the Mosquito® LCP (TTP labtech) system. The high-oligomer
fraction of BcTSPO never crystallized with this method; however, the monomer-dimer fraction of
BcTSPO in its Apo state did crystallize well in two different conditions. Type 1 crystals grew
from 0.1 M sodium cacodylate, 5% w/v PGA –LM (poly- l-glutamic acid, low molecular weight~
200-400 kDa), 30% v/v PEG 550MME (Polyethylene glycol monomethyl ether 550), pH 6.5;
these crystals gave data set: Apo Type1 monomer. Type 2 crystals grew from 0.2 M
ammonium sulfate, 0.1 M BIS-TRIS pH 5.5, 25% w/v polyethylene glycol 3,350; these crystals
gave data set: Apo Type2 monomer. Both conditions produced high quality crystals that
generally diffracted beyond 2.0 Å spacings. We were also able crystallize the complex of
BcTSPO with PK11195 in LCP in the condition of 3% PEG 4000, 0.066 sodium chloride, 0.02
Tris, pH 7.5; these crystals diffracted to 3.5 Å spaicngs and produced data set: PK11195 dimer.
For SAD phasing to obtain the first structure, Type 1 crystals were soaked in a saturated
solution of iodine in paraffin oil for seven hours prior to data collection.
Data collection and reduction
Data sets reported here were all collected at Brookhaven NSLS X4 beamlines. The data
set for iodine SAD phasing was collected on beamline X4A at 6 keV with a helium path between
the crystal and detector. These data were collected with 40 sec. exposure for 1° oscillation
frames. Inverse beam geometry was used in 30° wedges over a full 360° span. Data sets: Apo
Type 1 monomer, Apo Type 2 monomer, Apo dimer and PK11195 dimer were all measured on
X4C at 12.66 keV. These data were collected in 1° oscillation frames of 20-30 second x-ray
exposure each for 360° total span. The diffraction data were processed with both XDS (39) and
HKL2000 (HKL Research).
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Experimental phasing
Extensive molecular replacement attempts were made using the reported NMR structure
of mouse MmTSPO, but these were never successful. Because the LCP crystal diffracted quite
well, and we had been quite successful with sulfur phasing using the NSLS beamline NSLS
X4A, we tried to solve the BcTSPO crystal structure by sulfur SAD phasing. We collected more
than 30 data sets, and made some progress in substructure determination, but we were unable
to obtain adequate phasing for structure solution. After finally solving the structure, we realized
we had corrected located two sulfur atoms. The high background caused by monoolein
increased the difficulty in detecting anomalous signals. We also tried several heavy atoms;
some destroyed the crystals, and some gave anomalous signals but not strong enough for
phasing. From our experience, we knew iodine could react with cysteine and tyrosine or simply
be located to hydrophobic pockets in membrane protein crystals. Thus, we also tried SAD
phasing with iodine. 2 µL of saturated iodine in paraffin oil was added to an LCP drop (volume
about 1µL) that contained BcTSPO crystals. At different time intervals, we picked up crystals to
examine the diffraction and found that crystals soaked for seven hours in the iodine-paraffin oil
gave the best anomalous signal. With three data sets merged together we were able to
determine the sub-structure of three iodine atoms using SHELX C/D/E (40). Then, using
programs from the CCP4 package (41) for phase determination and ARP/wARP (42) for
automated model building, the initial BcTSPO crystal structure was obtained.
Structure determination and refinement
The initial iodo structure, including all residues of full-length BcTSPO, was refined using
Phenix 1.9 (43). The type 1 apo structure was completed by isomorphic variation, and the other
three crystal structures were solved by molecular replacement with Phaser (44) or CaspR (45)
based on the initial BcTSPO structure. Refinements and model building were performed
iteratively with Phenix 1.9 and Coot. 7.2 (46). Statistics for data collection and refinement are
presented in supplementary Table S1 and S2, respectively. In addition to the five structures
described in these tables, we also solved a type 2 apo structure in the presence of DMSO. That
structure is deposited with PDB code 4RYR.
Activity assays
UV-VIS method. Protoporphyrin (PpIX) has a prominent Soret band feature peaked at
410nm and weaker Q band features with four peaks at 628 nm, 574 nm, 537 nm and 503 nm
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individually. Ginter et al. (12) reported that TSPO was able to degrade PpIX. After reaction, the
product gave an observable blue color. We can monitor the decay of the Soret band and Q
bands and the appearance of the blue color to determine the PpIX degradation activity of
BcTSPO. UV-VIS scans of the solution mixtures of BcTSPO and PpIX showed two new peaks,
one at ~344 nm and another at ~570nm.
Fluorescence method. PpIX displays characteristic fluorescence emission at 632 nm
wavelength peak when excited at 405nm wavelength light. PpIX is a light and oxygen sensitive
chemical. When exposed to strong light and oxygen, PPIX undergoes a self-sensitized photo-
oxidation that yields a mixture of PPIX derived chemicals, photo-protoporphyrins (47). However,
self-sensitized photo-oxidation of PpIX is not observable on exposure to weak light and oxygen,
even for several hours. This is an important basis on which we designed our experiments.
We used the Fluoro Max-3 from Horiba Jobin Yvon to perform all the fluorescence spectra
experiment. Experiments with this instrument were conducted as a series of pulses, each of
which comprised 50 sec continuous illumination while recording a spectrum and 10 sec in the
dark while reading out the data. We used a 3mm square quartz cuvette. Reaction solutions
had a total volume of 230 µL prepared as follows: 1 µL saturated PpIX in DMSO was added to
199 µL reaction buffer (Buffer E), this mixture was then diluted 4X by adding 50 µL of this
mixture to 150 µL reaction buffer, and 30 µL protein solution at 1 mg/mL was added. For
controls, sample buffer replaced the proteins solution.
In our experiments we used three levels of excitation at 405nm wavelength: low-level, mid-
level, and high-level. We defined the light levels by instrument slit configurations: low-level,
excitation slit 0.5nm and emission slit 2nm; mid-level, excitation slit 2.5nm and emission slit
0.4nm; high-level, excitation slit 5.0nm and emission slit 2nm. Low-level excitation did not lead
to observable self-sensitized photo-oxidation as shown in Figs. 3D and S8A. High-level
excitation did generate secondary excited states (fig. S8B) as also observed with certain
BcTSPO mutants (fig. S8, C and D). We have two experimental controls: I) free PpIX in
reaction solution with 0.05% DDM exposed to low-level light for a series of pulses and II) PpIX
and bovine serum albumin (BSA) in reaction solution with 0.05% DDM exposed to low-level light
for series of pulses. (Fig. 3 D)
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Figure S1. Structure-based alignment of TSPO sequences. Amino acid sequences are given in the one-letter code and are identified by species abbreviations: Hs, Homo sapiens (man); Mm, Mus muscalis (mouse); Xt, Xenopus tropicalis (frog); Pp, Physcomitrella paten (moss); Rs, Rhodobacter sphaeroides (gram-negative bacterium); and Bc, Bacillus cereus (gram-positive bacterium). Residues that are identical in four or more of these proteins are colored red. Residues in helices in the structure of BcTSPO are indicated by solid bars.
TM1 α1,2
HsTSPO1 1----------------------------MAPPWVPAMGFTLAPSLGCFVGSRFVHGEGLRWYAGL 37 MmTSPO1 1----------------------------MPESWVPAVGLTLVPSLGGFMGAYFVRGEGLRWYASL 37 XtTSPO 1-----------------------------MPSWAPAIGLTILPHVGGIAGGLITRQEVKTWYTTL 36 HsTSPO2 1-------------------------------MRLQGAIFVLLPHLGPILVWLFTRDHMSGWCEGP 34 PpTSPO1 39---------------------------AKKPGVPSLIVACALPLAAGFLVSMFASPD--QWYKNL 74 RsTSPO 1---------------------------MMNMDWALFLTFLAACGAPATTGALLKPDE---WYDNL 35 BcTSPO 1------------------------------MFMKKSSIIVFFLTYGLFYVSSVLFPIDRTWYDAL 35
TM2 TM3
HsTSPO1 QKPSWHPPHWVLGPVWGTLYSAMGYGSYLVWKELGGFT-EKAVVPLGLYTGQLALNWAWPPIFFGARQ 104 MmTSPO1 QKPSWHPPRWTLAPIWGTLYSAMGYGSYIVWKELGGFT-EDAMVPLGLYTGQLALNWAWPPIFFGARQ 104 XtTSPO VKPSWRPPNWMFGPVWTTLYTSMGYGSYLIYKELGGLN-ENAVVPLGLYASQLALNWAWTPIFFGAHK 103 HsTSPO2 RMLSWCPFYKVLLLVQTAIYSVVGYASYLVWKDLGGGLGWPLALPLGLYAVQLTISWTVLVLFFTVHN 102 PpTSPO1 NKPSWTPPGPLFGLIWTFIYPVMGLASWLVWAD-GGFQ--RNGFALGAYFVQLGLNLLWSVLFFKFHS 139 RsTSPO NKPWWNPPRWVFPLAWTSLYFLMSLAAMRVAQLEGS------GQALAFYAAQLAFNTLWTPVFFGMKR 97 BcTSPO EKPSWTPPGMTIGMIWAVLFGLIALSVAIIYNNYGF----KPKTFWFLFLLNYIFNQAFSYFQFSQKN 99
TM4 TM5
HsTSPO1 MGWALVDLLLVSGAAAATTVAWYQVSPLAARLLYPYLAWLAFATTLNYCVWRDNHGWRGGRRLPE--- 169 MmTSPO1 MGWALADLLLVSGVATATTLAWHRVSPPAARLLYPYLAWLAFATVLNYYVWRDNSGRRGGSRLPE--- 169 XtTSPO IGWGLVDLLLLWGAAAATTISWYPISRPAAYLMLPYLAWLTLASALNYRIWKDNKDKSE--------- 162 HsTSPO2 PGLALLHLLLLYGLVVSTALIWHPINKLAALLLLPYLAWLTVTSALTYHLWRDSLCPVHQPQPTEKSD 170 PpTSPO1 VTLAFVDILALGAAVFTTIGAFQPVNHIAANLMKIYFGWVVFASVLTASILMKNSRGGH--------- 198 RsTSPO MATALAVVMVMWLFVAATMWAFFQLDTWAGVLFVPYLIWATAATGLNFEAMRLNWNRPEARA------ 159 BcTSPO LFLATVDCLLVAITTLLLIMFSSNLSKVSAWLLIPYFLWSAFATYLSWTIYSIN-------------- 153
Figure Sdeterminconcentridentify thdimeric, peak whequilibriufraction rhigh oligshowing covalent purple cobefore iligand sin Fig. 1
S2. Aspectnation. (Aration on a She ~10.5 mLand the sho
hen re-chroum with there-run at higomer. (D) Ealmost entibonds as s
ontours at 4.nclusion oftructure suA.
ts of BcTSA) Typical gSuperdex 20L peak as a oulder near 1matographe
e ‘dimer/mongh concentraElution profirely 15-mL shown with .0 σ. (F) PKf the ligandperimposed
PO solutioel filtration 0TM 10-300 high oligom
15 mL as a ed on the snomer’ fractation, showile for the ‘dmonomer. Tyr32, Cys
K11195 comd is shown d. In E an
8
on propertieprofile fromGL column.
mer species,monomer. same columtion. (C) Eng primarilyimer/monom(E) Iodinat
s107 and Tmplex with
contouredd F, ribbon
es and elemm detergent-
The void vothe peak at (B) Elution
mn. This fraElution profy the 13.7-mmer’ fraction ion of BcTS
Tyr150. BijvBcTSPO.
d at 2.0 σ wn backbone
ments of c-extracted Bolume is at ~~13.7 mL a
profile for thaction seemfile for the
mL dimer anre-run at lo
SPO. Iodinevoet-differenThe Fo-Fc with the fin
e structures
crystal strucBcTSPO at ~7.5 mL; anas being primhe ‘high oligoms not to b‘dimer/monod some ~9.
ow concentra reacted to
nce peaks adifference
nally interprs are colore
cture high
nd we marily omer’ be in omer’ 5-mL ation, form
are in map
reted ed as
Figure Scrystal scomprisinas a lighwith colo
S3. Stereostructure ong residues
ht blue 3D moring C (grey
odiagram oof BcTSPO F136 - I149
mesh contouy), N (blue) a
f the electat 1.7 Å
9. The electured at 1.5 σand O (red).
9
tron densityresolution.ron density dσ. The fitted
y distributi. The segmdistribution i
d model is s
ion from thment pictureis from a 2Fhown in stic
he Apo Tyed is from
Fo-Fc map sck represent
ype 2 TM5, hown tation
Figure Spotential degrees Panels Ecyan denfront viewabout thethe potenfor paneelectrosta
S4. Surfacat the molof blue an
E-F show thenote extents w of the apoe vertical axntial functionels E-F areatic coloring
ce features ecular surfad red deno
e residue conof sequenc
o monomer, is. Panel Cn for the dime identical t changes.
of BcTSPOace as calcuote variationnservation a
ce conservatas in Fig. 1
is for the Pmer as vieweto those fo
10
O structureulated with
ns in positivas calculatedtion and vari1C, and B isK11195 dim
ed from aboor A-D, only
es. Panels PDB2PQR
ve and negad by ConSuriability, resps for the rev
mer, orientedve (upper) ay the subs
A-D show server and
ative potentrf (50); degrepectively. Pverse side ad as in Fig. 1and below (lstitution of
the electroAPBS (48,
tial, respectees of purplePanel A is fofter 180º rot1G, and D slower). Surfconservatio
static , 49); tively. e and or the tation hows faces n for
A
B
11
Figure Swith oth(PDB: 2Mfrom Mm1A. Whealignmensuperposarrows dComparissuperimpsuperimpappreciasubstitutiis more ras in Fconservashowing BcTSPOprimarily primarily
S5. Comparher atomic-lMGY). The
mTSPO1 (cyaen individuant, residues sition. This idirect the inson to a crposable resposed onto bly from thion is readilyrepresentativig. 2B for
ation is lost iradically d
. (D) Compinvolves TMinvolves TM
risons of thevel TSPO Cα atoms an) were supal helices TM
that are ins highlightendicated Bcrystal structusidues were
RsTSPO (hat of RsTy accommodve. (C) CoBcTSPO an the MmTS
different ligaparison of BM2 helices aM1 and TM3
he BcTSPO structuresof 32 residuperimposed M1, TM3 or side in BcTd here by c
cTSPO sideure of RsTSe aligned. (cyan). ThTSPO A139dated in BcTomparison oand superimSPO1 modeland conformBcTSPO andand buries 8helices and
12
crystal stru. (A) Compues within 2onto those oTM4 were t
TSPO are pcomparing see chain ontoSPO A139T
BcTSPO he structure9T (PDB:4UTSPO, we sf cross-sect
mposed as . The PK11
mations andd RsTSPO d831 Å2 of su is more inti
ucture (apoparison to th2Å (46-71 ofof BcTSPOthen superim
placed outsielected conso that in th
T (PDB:4UC(black) is
e of WT RsUC1) (25); suggest thationed viewsin (A) for 195 ligand i
d poses in dimers. Theurface area.mate, buryin
o TSPO at 1e NMR modf TM2 and 1(black), andmposed baside based oserved residhe MmTSP
C1). The Cviewed as sTSPO (PDhowever,
t the A139T s of the ligan
MmTSPO1is included iMmTSPO1
e dimer interf. The interfng 1263 Å2 o
1.7Å resoludel of MmTS135-150 of
d viewed as ised on sequon the TM2dues in full.O1 model.
Cα atoms ofin Fig. 1A
DB:4UC3) dsince the mutant stru
nd-binding po. Pocket-n each cut-a compared face for BcTface for RsTof surface ar
ution) SPO1 TM5) in Fig.
uence 2/TM5
Red (B)
f 126 A as differs A→T
ucture ocket lining
away, with
TSPO TSPO rea.
Figure Sstructurethe electstructure
S6. Ligand e (red sphererostatic pote
e. Side chain
A
C
binding poes) in the apential surfaces are shown
ocket in Bcpo Type 2 me. (C) Steren for residue
13
cTSPO. (A) model (PDB:eoview of Ppes within van
B
Hydrogen-b4RYQ). (B
pIX docked in der Waals
bonded (dasB) Model of Pinto the apo contact dista
shed lines) wPpIX dockedType 2 BcT
ance to PpIX
water d into TSPO X.
14
Figure S7. Chemical structures of protoporphyrin IX (PpIX) and derivative compounds. Photo-oxidation yields multiple products; in aqueous micelles, this is a mixture of formyl substitutions either one or both of the vinyl groups (28). Biliverdin and phycocyanin are produced by enzymatic oxidative cleavage at the indicated methene bridge. The immediate substrate for the reaction producing biliverdin is heme (Fe-PpIX) and that for producing phycocyanobilin is biliverdin, derived from heme. The chemical structure of the bilindigin product of TSPO-mediated cleavage of PpIX is not known; however, by virtue of spectral similarities to biliverdin and phycocyanobilin (Fig. 3B) and relationship to the photo-oxidation of free PpIX, we speculate that one possible structure for bilindigin may be as shown.
Figure and whspectra fluorescIlluminatfluoresc(C and measure632 nm and 673during re
S8. Fluorhen associ
for free Pence emistion in a hence and gD) Fluoresced after the
primary-flu3 nm. Each eadout. (D
rescence eiated with
PpIX. (A) Ilssions at high level generation ocence spece indicated uorescencepulse com) After grad
excitations a mutantlumination 632 nm aof light le
of new featctra of PpIXsuccession
e peak andprised 50 sdual chang
15
s in protopt BcTSPO in a low
and 700 nads to timures, promX associaten of low-lev
d emergencsec. exposues through
porphyrin protein.
level of lignm, indepeme-depende
inently at 6ed with BcTvel light puce of exciteure during ah 39 pulses
IX compa(A and B
ght stably eendent of ent decay 673 nm andTSPO W51ulses showeed-state pea scan and s, incubatio
ared when B) Fluoresc
excites priduration. of the prim
d also at 649F. (C) Spe
ed decay oeaks at 649
10 sec. of n in the da
free ence mary (B) mary 9 nm. ectra
of the 9 nm dark
ark in
16
presence of PK11195 completely reversed the spectrum to the ground state. (E) Oscillation in fluorescence features of BcTSPO A142T. We contemplate that oscillations result for reduced TSPO affinity for excited states of PpIX and ground-state restoration when dissociated into solution.
17
18
Figure S9. Fluorescence analysis of TSPO-mediated activity in PpIX degradation and modulation. (A and B) Fluorescence analysis of BcTSPO W51F/W138F activity toward PpIX. Spectra after indicated light exposures are shown in (A), and time courses of 632-nm primary excited-state and 673-nm secondary excited-state fluorescences are tracked in (B). The 632-nm fluorescence for the double mutant decays more slowly than for either single mutant. Unlike W138F, here the 673-nm fluorescence appears and there is no bilindigin product; unlike W51F, there is no 649-nm fluorescence. (C and D) Fluorescence analysis of BcTSPO A142T activity toward PpIX. The changes for this mutant were reversed even without incubation in the dark and oscillatory behavior ensued (fig. S8E). We contemplate that oscillatory behavior may result from reduced TSPO affinity for excited states of PpIX and ground-state restoration when dissociated into solution. (E and F) Fluorescence analysis of WT XtTSPO activity toward PpIX. This degradation was irreversible in that incubation in that darkness did not restore the basal fluorescence, and the decay was entirely blocked when in saturated PK11195. We observed some instability of XtTSPO, so partial inactivity might have lowered the rate of reaction. (G and H) Fluorescence analysis of HsTSPO1 A147T activity toward PpIX. When this reaction was performed in saturated PK11195, we observed inhibition of the reaction followed by reversal and then small-scale oscillations, as we also saw for BcTSPO A142T. (I and J) Fluorescence analysis of WT HsTSPO2 activity toward PpIX. This non-mitochondrial paralog HsTSPO2 also carries the A→T change, analogous with HsTSPO1 A147T, and it additionally lacks the catalytically critical analog of Trp51 of BcTSPO (fig. S1). PK11195 inhibited the reaction, but not as much as with HsTSPO1 A147T. We expect that the predominant Ala147 polymorph of HsTSPO1 will degrade PpIX since it shares identical catalytic components with BcTSPO and XtTSPO, whereas HsTSPO1 A147T and HsTSPO2 are variants and do not yield degraded product. Spectra and time courses for each pair, (A and B) through (I and J), are displayed as in (A) and (B), except that there is no 673-nm fluorescence to follow in (F).
Figure Sligand-bimodeled positionscomplex.
S10. Disposnding pockeas in Fig. S
s to PpIX at.
A
B
sition of conet of BcTSPS6C. Dottedtoms. (B)
nserved trypPO. (A) Sted lines show Detail of try
19
ptophan resiereodrawing
closest appyptophan Nδ
idues relativof the apo
proaches of δ interaction
ve to PpIX mType 2 str
candidate trns with PpI
modeled intucture with ryptophan raX in the do
o the PpIX
adical ocked
20
Table S1. Diffraction data statistics _____________________________________________________________________________________ Dataset Iodo Type1 monomer Apo Type1 monomer Apo Type2 monomer Apo dimer PK11195 dimer _____________________________________________________________________________________________________________________
Beamline NSLS X4A NSLS X4C NSLS X4C NSLS X4C NSLS X4C λ (Å) 2.0735 0.9791 0.9791 0.9791 0.9791 Space group P212121 P212121 P212121 P21 P21 Unit cell dimensions a, b, c (Å) 33.36, 49.54, 99.21 33.61, 49.37, 97.85 28.84, 54.64, 107.37 33.98, 104.93, 53.86 56.66, 54.55,58.56 Za
a 1 1 1 2 2
Solvent content (%) 36.7 36.1 38.1 44.0 40.0 Bragg spacings (Å) 40‐2.80 (2.87‐2.80) 49‐2.01 (2.06‐2.01) 40‐1.70 (1.73‐1.70) 18‐4.10 (4.21‐4.10) 50‐3.50 (3.56‐3.50) Total reflections 287312 160773 166478 10424 13556 Unique reflections 4153 11360 17687 5345 4245 Multiplicity 69.2(31.6) 14.2 (11.6) 6.1 (4.4) 1.95 (1.71) 3.2 (2.5) Completenessb (%) 94.7 (70.5) 99.4 (95.8) 93.4 (91.1) 94.3 (91.2) 96.2(88.6) Rmerge
c 0.104 (1.11) 0.231 (3.06) 0.251 0.176 Rpim
d 0.013 (0.197) 0.065 (0.956) 0.065 (0.754) 0.113 (0.575) Rmeas
e 0.113 (1.14) 0.247 (3.33) 0.186 (>1) 0.356 (1.20) 0.209 (0.983) CC1/2 (%)
f 100 (91.8) 99.8 (34.8) 91.5 (49.8) 99.1 (61.2) 80.0 (69.9) CCanom 88.2 (8.7) <I/σ(I)>g 42.6(3.3) 11.7 (0.9) 13.5 (0.72) 1.63 0.55) 4.35 (0.63) Anomalous completeness 94.8 (69.3) Anomalous multiplicity 38.4 (16.4) _____________________________________________________________________________________________________________________ a Za stands for number of subunits per asymmetric unit. b Values in the outermost shell are given in parentheses. c Rmerge = (Σ |Ii − < Ii > |) / Σ |Ii|, where Ii is the integrated intensity of a given reflection. e Rmeas is the redundancy‐independent merging R factor (51) d Rpim is precision‐indicating and multiplicity‐weighted Rmerge. f CC½ is the correlation coefficient of integrated intensities between randomly split two half data sets (52) g <I/σ(I)> = <(<Ii>) / <σ(<Ii>)>
Table S2. Refinement statistics _____________________________________________________________________________________ Structure Iodo Type1 monomer Apo Type1 monomer Apo Type2 monomer Apo dimer PK11195 dimer _____________________________________________________________________________________________________________________
Resolution (Å) 2.8 2.0 1.7 4.1 3.5 Unique reflections 4122 11301 17505 2868 4221 Total atoms 1259 1450 1431 2444 2548 Protein atoms 1256 141 1335 2444 2548 Iodine atoms 3 Water molecules 0 27 95 0 0 Rwork
a 0.217 0.208 0.224 0.345 0.236
Rfreeb 0.272 0.252 0.247 0.374 0.314
RMS bond (Å) 0.007 0.002 0.006 0.005 0.005 RMS angle (Å) 0.768 0.655 0.890 1.068 0.898 Average B factor (Å) 62.5 36.1 22.0 125.0 74.9 Ramachandran analysisc favored/allowed (%) 99.3/0.7 99.3/0.7 98.0/2.0 98.7/1.3 98.3/1.7 PDB code 4RYM 4RYN 4RYQ 4RYJ 4RYI _____________________________________________________________________________________________________________________ a Rwork = (Σ | |Fo| − |Fc| |) / Σ|Fo|, where Fo and Fc denote observed and calculated structure factors, respectively. bRfree was calculated using 5% of data excluded from refinement. cMolprobity (53).
References and Notes
1. V. Papadopoulos, M. Baraldi, T. R. Guilarte, T. B. Knudsen, J.-J. Lacapère, P. Lindemann, M.
D. Norenberg, D. Nutt, A. Weizman, M.-R. Zhang, M. Gavish, Translocator protein
(18kDa): New nomenclature for the peripheral-type benzodiazepine receptor based on its
structure and molecular function. Trends Pharmacol. Sci. 27, 402–409 (2006). Medline
doi:10.1016/j.tips.2006.06.005
2. C. Braestrup, R. F. Squires, Specific benzodiazepine receptors in rat brain characterized by
high-affinity (3H)diazepam binding. Proc. Natl. Acad. Sci. U.S.A. 74, 3805–3809 (1977).
Medline doi:10.1073/pnas.74.9.3805
3. A. A. Yeliseev, S. Kaplan, A sensory transducer homologous to the mammalian peripheral-
type benzodiazepine receptor regulates photosynthetic membrane complex formation in
Rhodobacter sphaeroides 2.4.1. J. Biol. Chem. 270, 21167–21175 (1995). Medline
doi:10.1074/jbc.270.36.21167
4. A. A. Yeliseev, K. E. Krueger, S. Kaplan, A mammalian mitochondrial drug receptor
functions as a bacterial “oxygen” sensor. Proc. Natl. Acad. Sci. U.S.A. 94, 5101–5106
(1997). Medline doi:10.1073/pnas.94.10.5101
5. A. G. Mukhin, V. Papadopoulos, E. Costa, K. E. Krueger, Mitochondrial benzodiazepine
receptors regulate steroid biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 86, 9813–9816
(1989). Medline doi:10.1073/pnas.86.24.9813
6. A. Verma, J. S. Nye, S. H. Snyder, Porphyrins are endogenous ligands for the mitochondrial
(peripheral-type) benzodiazepine receptor. Proc. Natl. Acad. Sci. U.S.A. 84, 2256–2260
(1987). Medline doi:10.1073/pnas.84.8.2256
7. V. Papadopoulos, W. L. Miller, Role of mitochondria in steroidogenesis. Best Pract. Res. Clin.
Endocrinol. Metab. 26, 771–790 (2012). Medline doi:10.1016/j.beem.2012.05.002
8. N. Rosenberg, O. Rosenberg, A. Weizman, L. Veenman, M. Gavish, In vitro catabolic effect
of protoporphyrin IX in human osteoblast-like cells: Possible role of the 18 kDa
mitochondrial translocator protein. J. Bioenerg. Biomembr. 45, 333–341 (2013). Medline
doi:10.1007/s10863-013-9501-4
9. V. Papadopoulos, H. Amri, N. Boujrad, C. Cascio, M. Culty, M. Garnier, M. Hardwick, H. Li,
B. Vidic, A. S. Brown, J. L. Reversa, J. M. Bernassau, K. Drieu, Peripheral
benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids 62, 21–28
(1997). Medline doi:10.1016/S0039-128X(96)00154-7
10. L. N. Tu, K. Morohaku, P. R. Manna, S. H. Pelton, W. R. Butler, D. M. Stocco, V. Selvaraj,
Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable
with no effects on steroid hormone biosynthesis. J. Biol. Chem. 289, 27444–27454
(2014). Medline doi:10.1074/jbc.M114.578286
11. G. Wendler, P. Lindemann, J. J. Lacapère, V. Papadopoulos, Protoporphyrin IX binding and
transport by recombinant mouse PBR. Biochem. Biophys. Res. Commun. 311, 847–852
(2003). Medline doi:10.1016/j.bbrc.2003.10.070
12. C. Ginter, I. Kiburu, O. Boudker, Chemical catalysis by the translocator protein (18 kDa).
Biochemistry 52, 3609–3611 (2013). Medline doi:10.1021/bi400364z
13. L. Veenman, M. Gavish, W. Kugler, Apoptosis induction by erucylphosphohomocholine via
the 18 kDa mitochondrial translocator protein: Implications for cancer treatment.
Anticancer. Agents Med. Chem. 14, 559–577 (2014). Medline
doi:10.2174/1871520614666140309230338
14. F. M. Lartey, G. O. Ahn, B. Shen, K. T. Cord, T. Smith, J. Y. Chua, S. Rosenblum, H. Liu,
M. L. James, S. Chernikova, S. W. Lee, L. J. Pisani, R. Tirouvanziam, J. W. Chen, T. D.
Palmer, F. T. Chin, R. Guzman, E. E. Graves, B. W. Loo Jr., PET imaging of stroke-
induced neuroinflammation in mice using [18
F]PBR06. Mol. Imaging Biol. 16, 109–117
(2014). Medline doi:10.1007/s11307-013-0664-5
15. T. Zhou, Y. Dang, Y.-H. Zheng, The mitochondrial translocator protein, TSPO, inhibits HIV-
1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein
degradation pathway. J. Virol. 88, 3474–3484 (2014). Medline doi:10.1128/JVI.03286-13
16. T. Ruksha, M. Aksenenko, V. Papadopoulos, Role of translocator protein in melanoma
growth and progression. Arch. Dermatol. Res. 304, 839–845 (2012). Medline
doi:10.1007/s00403-012-1294-5
17. W. C. Kreisl, C. H. Lyoo, M. McGwier, J. Snow, K. J. Jenko, N. Kimura, W. Corona, C. L.
Morse, S. S. Zoghbi, V. W. Pike, F. J. McMahon, R. S. Turner, R. B. Innis; Biomarkers
Consortium PET Radioligand Project Team, In vivo radioligand binding to translocator
protein correlates with severity of Alzheimer’s disease. Brain 136, 2228–2238 (2013).
Medline doi:10.1093/brain/awt145
18. X. Qi, J. Xu, F. Wang, J. Xiao, Translocator protein (18 kDa): A promising therapeutic target
and diagnostic tool for cardiovascular diseases. Oxid. Med. Cell. Longev. 2012, 162934
(2012). Medline doi:10.1155/2012/162934
19. R. Rupprecht, V. Papadopoulos, G. Rammes, T. C. Baghai, J. Fan, N. Akula, G. Groyer, D.
Adams, M. Schumacher, Translocator protein (18 kDa) (TSPO) as a therapeutic target for
neurological and psychiatric disorders. Nat. Rev. Drug Discov. 9, 971–988 (2010).
Medline doi:10.1038/nrd3295
20. F. Delavoie, H. Li, M. Hardwick, J.-C. Robert, C. Giatzakis, G. Péranzi, Z.-X. Yao, J.
Maccario, J.-J. Lacapère, V. Papadopoulos, In vivo and in vitro peripheral-type
benzodiazepine receptor polymerization: Functional significance in drug ligand and
cholesterol binding. Biochemistry 42, 4506–4519 (2003). Medline
doi:10.1021/bi0267487
21. D. R. Owen, A. J. Yeo, R. N. Gunn, K. Song, G. Wadsworth, A. Lewis, C. Rhodes, D. J.
Pulford, I. Bennacef, C. A. Parker, P. L. StJean, L. R. Cardon, V. E. Mooser, P. M.
Matthews, E. A. Rabiner, J. P. Rubio, An 18-kDa translocator protein (TSPO)
polymorphism explains differences in binding affinity of the PET radioligand PBR28. J.
Cereb. Blood Flow Metab. 32, 1–5 (2012). Medline doi:10.1038/jcbfm.2011.147
22. R. Mizrahi, P. M. Rusjan, J. Kennedy, B. Pollock, B. Mulsant, I. Suridjan, V. De Luca, A. A.
Wilson, S. Houle, Translocator protein (18 kDa) polymorphism (rs6971) explains in-vivo
brain binding affinity of the PET radioligand [18
F]-FEPPA. J. Cereb. Blood Flow Metab.
32, 968–972 (2012). Medline doi:10.1038/jcbfm.2012.46
23. V. M. Korkhov, C. Sachse, J. M. Short, C. G. Tate, Three-dimensional structure of TspO by
electron cryomicroscopy of helical crystals. Structure 18, 677–687 (2010). Medline
doi:10.1016/j.str.2010.03.001
24. Ł. Jaremko, M. Jaremko, K. Giller, S. Becker, M. Zweckstetter, Structure of the
mitochondrial translocator protein in complex with a diagnostic ligand. Science 343,
1363–1366 (2014). Medline doi:10.1126/science.1248725
25. F. Li, J. Liu, Y. Zheng, R. M. Garavito, S. Ferguson-Miller, Crystal structures of translocator
protein (TSPO) and mutant mimic of a human polymorphism. Science 347, 555–558
(2015).
26. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D,
Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q,
Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
27. A. Marcelli, I. Jelovica Badovinac, N. Orlic, P. R. Salvi, C. Gellini, Excited-state absorption
and ultrafast relaxation dynamics of protoporphyrin IX and hemin. Photochem.
Photobiol. Sci. 12, 348–355 (2013). Medline doi:10.1039/c2pp25247c
28. S. Jockusch, C. Bonda, S. Hu, Photostabilization of endogenous porphyrins: Excited state
quenching by fused ring cyanoacrylates. Photochem. Photobiol. Sci. 13, 1180–1184
(2014). Medline doi:10.1039/C4PP00090K
29. G. S. Cox, D. G. Whitten, Mechanisms for the photooxidation of protoporphyrin IX in
solution. J. Am. Chem. Soc. 104, 516–521 (1982). doi:10.1021/ja00366a023
30. J. Dalton, C. A. McAuliffe, D. H. Slater, Reaction between molecular oxygen and photo-
excited protoporphyrin IX. Nature 235, 388 (1972). Medline doi:10.1038/235388a0
31. R. Pogni, M. C. Baratto, C. Teutloff, S. Giansanti, F. J. Ruiz-Dueñas, T. Choinowski, K.
Piontek, A. T. Martínez, F. Lendzian, R. Basosi, A tryptophan neutral radical in the
oxidized state of versatile peroxidase from Pleurotus eryngii: A combined
multifrequency EPR and density functional theory study. J. Biol. Chem. 281, 9517–9526
(2006). Medline doi:10.1074/jbc.M510424200
32. W. Frank, K. M. Baar, E. Qudeimat, M. Woriedh, A. Alawady, D. Ratnadewi, L. Gremillon,
B. Grimm, R. Reski, A mitochondrial protein homologous to the mammalian peripheral-
type benzodiazepine receptor is essential for stress adaptation in plants. Plant J. 51,
1004–1018 (2007). Medline doi:10.1111/j.1365-313X.2007.03198.x
33. J. C. Koningsberger, B. S. Van Asbeck, J. Van Hattum, L. J. J. M. Wiegman, G. P. Van
Berge Henegouwen, J. J. M. Marx, The effect of porphyrins on cellular redox systems: A
study on the dark effect of porphyrins on phagocytes. Eur. J. Clin. Invest. 23, 716–723
(1993). Medline doi:10.1111/j.1365-2362.1993.tb01291.x
34. J.-A. Farrera, A. Jaumà, J. M. Ribó, M. A. Peiré, P. P. Parellada, S. Roques-Choua, E.
Bienvenue, P. Seta, The antioxidant role of bile pigments evaluated by chemical tests.
Bioorg. Med. Chem. 2, 181–185 (1994). Medline doi:10.1016/S0968-0896(00)82013-1
35. M. Punta, J. Love, S. Handelman, J. F. Hunt, L. Shapiro, W. A. Hendrickson, B. Rost,
Structural genomics target selection for the New York consortium on membrane protein
structure. J. Struct. Genomics 10, 255–268 (2009). Medline doi:10.1007/s10969-009-
9071-1
36. R. Bruni, B. Kloss, High-throughput cloning and expression of integral membrane proteins in
Escherichia coli. Curr. Protoc. Protein Sci. 74, 29.6.1–29.6.34 (2013). Medline
37. A. Cheng, B. Hummel, H. Qiu, M. Caffrey, A simple mechanical mixer for small viscous
lipid-containing samples. Chem. Phys. Lipids 95, 11–21 (1998). Medline
doi:10.1016/S0009-3084(98)00060-7
38. M. Caffrey, V. Cherezov, Crystallizing membrane proteins using lipidic mesophases. Nat.
Protoc. 4, 706–731 (2009). Medline doi:10.1038/nprot.2009.31
39. W. Kabsch, XDS. Acta Crystallogr. D 66, 125–132 (2010). Medline
doi:10.1107/S0907444909047337
40. G. M. Sheldrick, Experimental phasing with SHELXC/D/E: Combining chain tracing with
density modification. Acta Crystallogr. D 66, 479–485 (2010). Medline
doi:10.1107/S0907444909038360
41. M. D. Winn, C. C. Ballard, K. D. Cowtan, E. J. Dodson, P. Emsley, P. R. Evans, R. M.
Keegan, E. B. Krissinel, A. G. Leslie, A. McCoy, S. J. McNicholas, G. N. Murshudov, N.
S. Pannu, E. A. Potterton, H. R. Powell, R. J. Read, A. Vagin, K. S. Wilson, Overview of
the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).
Medline doi:10.1107/S0907444910045749
42. G. Langer, S. X. Cohen, V. S. Lamzin, A. Perrakis, Automated macromolecular model
building for x-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171–
1179 (2008). Medline doi:10.1038/nprot.2008.91
43. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd,
L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R.
Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, P. H. Zwart,
PHENIX: A comprehensive Python-based system for macromolecular structure solution.
Acta Crystallogr. D 66, 213–221 (2010). Medline doi:10.1107/S0907444909052925
44. R. J. Read, Pushing the boundaries of molecular replacement with maximum likelihood. Acta
Crystallogr. D 57, 1373–1382 (2001). Medline doi:10.1107/S0907444901012471
45. J.-B. Claude, K. Suhre, C. Notredame, J.-M. Claverie, C. Abergel, CaspR: A web server for
automated molecular replacement using homology modelling. Nucleic Acids Res. 32
(suppl. 2), W606–W609 (2004). Medline doi:10.1093/nar/gkh400
46. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta
Crystallogr. D 66, 486–501 (2010). Medline doi:10.1107/S0907444910007493
47. M. Krieg, D. G. Whitten, Self-sensitized photo-oxidation of protoporphyrin IX and related
porphyrins in erythrocyte ghosts and microemulsions: A novel photo-oxidation pathway
involving singlet oxygen. J. Photochem. 25, 235–252 (1984).
48. T. J. Dolinsky, J. E. Nielsen, J. A. McCammon, N. A. Baker, PDB2PQR: An automated
pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids
Res. 32 (suppl. 2), W665–W667 (2004). Medline doi:10.1093/nar/gkh381
49. N. A. Baker, D. Sept, S. Joseph, M. J. Holst, J. A. McCammon, Electrostatics of
nanosystems: Application to microtubules and the ribosome. Proc. Natl. Acad. Sci.
U.S.A. 98, 10037–10041 (2001). Medline doi:10.1073/pnas.181342398
50. H. Ashkenazy, E. Erez, E. Martz, T. Pupko, N. Ben-Tal, ConSurf 2010: Calculating
evolutionary conservation in sequence and structure of proteins and nucleic acids.
Nucleic Acids Res. 38 (suppl. 2), W529–W533 (2010). Medline doi:10.1093/nar/gkq399
51. K. Diederichs, P. A. Karplus, Improved R-factors for diffraction data analysis in
macromolecular crystallography. Nat. Struct. Biol. 4, 269–275 (1997). Medline
doi:10.1038/nsb0497-269
52. P. A. Karplus, K. Diederichs, Linking crystallographic model and data quality. Science 336,
1030–1033 (2012). Medline doi:10.1126/science.1218231
53. V. B. Chen, W. B. Arendall 3rd, J. J. Headd, D. A. Keedy, R. M. Immormino, G. J. Kapral,
L. W. Murray, J. S. Richardson, D. C. Richardson, MolProbity: All-atom structure
validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).
Medline doi:10.1107/S0907444909042073