(fig. S5C). We also compared Bc TSPO to recent
structures of R. sphaeroides TSPO (Rs TSPO) (25)
and here found substantial similarity. On superimposition of those 126 of 148 Ca atoms within
3 Å of one another for Bc TSPO versus Rs TSPO
Ala139→Thr139 (A139T) (26) (PDB IDs 4RYQ versus 4UC1), the RMSD value was 1.17 Å, with TM1
helices most disparate (fig. S5B). TM1 is the least
conserved helix (fig. S1), and it is at the dimer
interface of Rs TSPO but not at that of Bc TSPO
(fig. S5D).
Both protomers of the Bc TSPO-PK11195 dimer
showed omit-map density in the pocket opening
between TM1 and TM2 (fig. S2F). When fitted by
the ligand structure, the resulting model has the
carbonyl-oxygen atom of PK11195 hydrogen bonded
to indole-NH groups of both Trp51 and Trp138,
and the Cl atom of the ligand is in van der Waals
contact with Asn87 (Fig. 2A). PK11195 in Bc TSPO
also makes van der Waals contacts with residues
Ser22, Tyr32, Pro42, Ile47, Phe55, Phe90, Ser91, Gln94,
Cys107, Ala142, and Leu145. These contacts ema-
nate from each of the transmembrane helices plus
the TM1-TM2 loop. All among them except Ser22
are widely conserved residues. Because the apo
and ligated Bc TSPO proteins are essentially iso-
structural, the ligand-binding site is a highly con-
served cavity in apo Bc TSPO (Fig. 2B). This cavity
is water-filled in our high-resolution apo struc-
tures (fig. S6A), and PpIX can be satisfactorily
docked into the cavity in a particular orientation
(fig. S6, B and C). The PK11195 conformation,
ligand binding pose, and protein contacts are very
different in the model for Mm TSPO1 (fig. S5A).
The side chain of Ala142 protrudes into the binding pocket such that its mutation would be expected to interfere with ligand binding. In accord,
recent reports show that TSPO radioligands PBR28
(21) and [18F]-FEPPA (22) have different binding
affinities for the predominant Ala147 and the minor
Thr147 polymorphic variants of human TSPO1 (
position 142 in Bc TSPO).
In attempting to bind PpIX to Bc TSPO, we no-
ticed that initially red mixtures turned blue on
standing out in the light for a few minutes, but not
when dark. Thus, considering the reported pho-
tooxidative degradation of PpIX by Chlorobium
tepidum TSPO (Ct TSPO) (12), we characterized
this activity of BcTSPO toward PpIX. As for
Ct TSPO, we observed a rapid decrease of the
Soret absorbance at 405 nm when in the light
(Fig. 3A) and a concomitant loss of the charac-
teristic PpIX fluorescence at 629 and 697 nm
(632 and 700 nm for Bc TSPO-PpIX) when illumi-
nated by ultraviolet (UV) light (27, 28). These
changes were irreversible, and saturated PK11195
substantially inhibited the PpIX decay but did
not entirely block it.
The post-illumination absorbance spectrum of
Bc TSPO-PpIX is like that reported from Ct TSPO-PpIX (12), and the spectrum that we extracted for
the photo-degradation product shows features like
those of spectra from biliverdin and phycocyanobilin, but with absorption peaks further blueshifted in series (Fig. 3B). By analogy with biliverdin
and for its indigo color, we call this product
bilindigin. Because the photooxidation of PpIX
yields oxidized vinyl groups, predominantly generating formyl groups when in aqueous micelles
Fig. 3. Spectral analysis of bacterial TSPO-mediated activity in PpIX degradation and modulation. (A) UV-visible
spectra of WT Bc TSPO with PpIX before and after reaction.
AU, absorbance units. (B) Comparison of the spectrum for
the bilindigin degradation product as extracted from the
postreaction spectrum in (A), with spectra from biliverdin
and phycocyanobilin. (C and D) Fluorescence analysis of
WT BcTSPO activity toward PpIX. Specta after indicated
light exposures are shown in (C), and time courses of
fluorescence at the 632-nm ground-state peak are tracked
in (D). Exposure time is measured in light pulses, where
each pulse comprised 50 s light plus 10 s read-out in the
dark. Time courses are compared for PpIX in the indicated
associations. (E and F) Fluorescence analysis of BcTSPO
W138F activity toward PpIX. Spectra and time courses are
as described for (C) and (D). (G and H) Fluorescence
analysis of Bc TSPO W51F activity toward PpIX. Spectra and
time courses are as described for (C) and (D). Error bars in
(D), (F), and (H) indicate SD.