Secondary and tertiary structures of the transmembrane domains of the translocator protein TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding

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Abstract

Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from 1H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using 1H–15N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.

Abbreviations

DPC
dodecyl phosphocholine
DIPEA
N,N-diisopropylethylamine
DSS
dimethylsilapentane-sulfonic acid, sodium salt
HATU
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium hexafluorophosphate
HSQC
heteronuclear single quantum correlation
NOESY
nuclear Overhauser enhancement spectroscopy
PAL-PEG-PS
5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeric acid-polyethylene glycol-polystyrene
TOCSY
Total correlation spectroscopy

Keywords

TSPO
PBR
NMR and CD spectroscopy
Drug ligand binding

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