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Submitted on July 30, 2004
Accepted on October 27, 2004
CEA-Saclay, DBJC/SBFM and URA CNRS 2096, Bat 532 91191 Gif sur Yvette Cedex, France; Unité INSERM U410, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75870 Paris Cedex 18, France; Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20057, USA
* To whom correspondence should be addressed. 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 (recPBR) into proteoliposomes demonstrated high affinity PK 11195 and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recPBR and NMR. 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. NMR 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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