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

Abstract

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.

Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface

“…Significant structural and operational differences, however, exist between the CDC cholesterol recognition motif and the CRAC motif. The CRAC motif (L/V-X (1-5) -Y-X (1-5) -R/K) is typically located adjacent to a transmembrane α-helix that positions this sequence within the nonpolar core of the bilayer where it can form contacts along the length of the cholesterol molecule (29). In contrast, the CDC Thr-Leu pair must recognize specific features of cholesterol at or near the membrane surface to initiate the cholesterol-dependent interaction of the CDC with the cell.…”

Identification of a Binding Motif in the S5 Helix That Confers Cholesterol Sensitivity to the TRPV1 Ion Channel

“…We consequently explored rTRPV1 for possible cholesterolbinding sites. One established sequence selective for binding cholesterol is the putative CRAC motif, first identified in the benzodiazepine receptor (43) and later identified in many other proteins and peptides (40,44,45). This motif is defined as a sequence pattern of -(Leu/Val-(X) 1-5 -Tyr-(X) 1-5 -Arg/Lys)-, in which (X) [1][2][3][4][5] represents between one and five residues of any amino acid (40,45).…”

Imaging of neuroinflammation

“…TSPO is directly or indirectly involved in many functions, including regulation of cholesterol transport [5], synthesis of steroid hormones [6] and apoptosis [7].…”