www.sciencemag.org/content/343/6221/551/suppl/DC1

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: wayne@xtl.cumc.columbia.edu

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

2  

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

3  

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

4  

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).

5  

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

6  

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)

 

7  

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).    

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