Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor.
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.
Jamin
Nadège
N
Département de Biologie Joliot Curie/Service de Biophysique des Fonctions Membranaires and Unité de Recherche Associée Centre National de la Recherche Scientifique 2096, Gif-sur-Yvette Cedex, France. jamin@dsvidf.cea.fr
Neumann
Jean-Michel
JM
Ostuni
Mariano A
MA
Vu
Thi Kim Ngoc
TK
Yao
Zhi-Xing
ZX
Murail
Samuel
S
Robert
Jean-Claude
JC
Giatzakis
Christoforos
C
Papadopoulos
Vassilios
V
Lacapère
Jean-Jacques
JJ
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D008024:Ligands; D008081:Liposomes / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008823:Micelles; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D018079:Receptors, GABA / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
D000596:Amino Acids; C463819:Bzrp protein, mouse; D002352:Carrier Proteins; D018076:DNA, Complementary; D008024:Ligands; D008081:Liposomes; D008823:Micelles; D010455:Peptides; D011522:Protons; D018079:Receptors, GABA; D011994:Recombinant Proteins; D002784:Cholesterol; D010196:Pancreatic Elastase; C017130:cholesterol-binding protein