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.
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 (1)H-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 (1)H-(15)N 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.
Murail
Samuel
S
Commissariat à l'Energie Atomique, Centre de Saclay, Institut de Biologie et Technologies de Saclay and URA CNRS 2096, Service de Bioénergétique Biologie Structurale et Mécanismes, Gif sur Yvette Cedex, France.
Robert
Jean-Claude
JC
Coïc
Yves-Marie
YM
Neumann
Jean-Michel
JM
Ostuni
Mariano A
MA
Yao
Zhin-Xing
ZX
Papadopoulos
Vassilios
V
Jamin
Nadège
N
Lacapère
Jean-Jacques
JJ
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D006801:Humans; D051379:Mice; D051336:Mitochondrial Membranes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D020836:Protein Structure, Quaternary / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D018079:Receptors, GABA / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
C463819:Bzrp protein, mouse; D018079:Receptors, GABA; D002784:Cholesterol