Protein structure. Structure and activity of tryptophan-rich TSPO proteins

Abstract

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 (BcTSPO) 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.

Figure 1. Structures of BcTSPO

(A and B) Ribbon drawing of TSPO from the type 1 apo structure as viewed from within the membrane plane (A) and from the periplasm above (B). Coloring progresses spectrally from the N-terminus (blue) to the C-terminus (red). Side chains are shown for conserved tryptophan residues W31, W40, W51 and W138. (C and D) Electrostatic potential at the molecular surface of the apo monomer as oriented in (A and B), respectively. Positive potentials are in degrees of blue; negatives are in red. Dotted planes show membrane boundaries computed by the OPM server. (E) Molecular surface as in (D) but colored by Consurf sequence conservation. Variable positions are in degrees of turquoise; conserved positions are in degrees of maroon. (F) Ribbon drawing of the apo dimer in detergent. Tryptophan residues and coloring are as in (A). (G) Ribbon drawing of the PK11195 dimer in LCP. The PK11195 moieties are drawn as pink sticks.

Figure 2. Features of the ligand-binding site in BcTSPO

(A) PK11195 in the binding pocket. The ligand is drawn as a stick figure with atom coloring of carbon (light grey), nitrogen (blue) and oxygen (red). Portions of a ribbon drawing of the TSPO protein structure are displayed faintly with coloring in Fig. 1(G). Side chains that contact PK11195 are drawn in stick representation and have carbon atoms colored as for the backbone. (B) Cross-sectioned views of the ligand-binding pocket. The surface of an apo monomer, oriented as in Fig. 1(A) and sliced by a vertical plane perpendicular to the page, was opened as a book to show both sides of the invaginated pocket. The planar cut surfaces are grey and exposed molecular surfaces and visible atomic structures are in ConSurf coloring as in Fig. 1(E).

Figure 3. Spectral analysis of bacterial TSPO-mediated activity in PpIX degradation and modulation

(A) UV-visible spectra of WT BcTSPO with PpIX before and after reaction. (B) Comparison of the spectrum for the bilindigin degradation product as extracted from post-reaction 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 sec. light plus 10 sec. 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 BcTSPO W51F activity toward PpIX. Spectra and time courses are as described for (C) and (D).