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Characterization of the Cholesterol Recognition Amino Acid Consensus Sequence of the Peripheral-Type Benzodiazepine Receptor

Commissariat à l’Energie Atomique-Saclay (N.J., J.-M.N., T.K.N.V., S.M.,), Département de Biologie Joliot Curie/Service de Biophysique des Fonctions Membranaires and Unité de Recherche Associée Centre National de la Recherche Scientifique 2096, 91191 Gif sur Yvette Cedex, France; Institut National de la Santé et de la Recherche Médicale Unité 410 (M.A.O., J.-C.R., J.-J.L.), Faculté de Médecine Xavier Bichat, 75870 Paris Cedex 18, France; and Department of Biochemistry and Molecular Biology (Z.-X.Y., C.G., V.P.), Georgetown University Medical Center, Washington, DC 20057

Address all correspondence and requests for reprints to: Dr. Nadège Jamin, Commissariat à l’Energie Atomique-Saclay, Département de Biologie Joliot Curie/Service de Biophysique des Fonctions Membranaires and Unité de Recherche Associée Centre National de la Recherche Scientifique 2096, Bâtiment 532, 91191 Gif-sur-Yvette Cedex, France. E-mail: jamin{at}dsvidf.cea.fr.

We previously defined a cholesterol recognition/interaction amino acid consensus sequence [CRAC: L/V–X (1–5)–Y–X (1–5)-R/K] in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane protein. This protein is involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. Reconstituted wild-type recombinant PBR into proteoliposomes demonstrated high-affinity 2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144–169) containing the CRAC domain indicated a helical conformation for the L144–S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria.

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