Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor
Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor
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DOI：
10.1210/me.2004-0308
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发表时间：
2005-03-01
影响因子：
--
通讯作者：
Lacapère, JJ
中科院分区：
文献类型：
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作者：
Jamin, N;Neumann, JM;Lacapère, JJ
我们先前在外周型苯二氮卓受体（PBR）的羧基末端定义了胆固醇识别/相互作用氨基酸共有序列[ CRAC：L/V-X（1 - 5）-Y-X（1 - 5）- R/ K]，所述外周型苯二氮卓受体（PBR）是存在于线粒体外膜蛋白中的高亲和力药物和胆固醇结合蛋白。这种蛋白质参与调节胆固醇转运到线粒体中，这是类固醇生物合成中的速率决定步骤。将野生型重组PBR重组到脂蛋白体中，显示出高亲和力的2-氯苯基）-N-甲基-N-（1-甲基丙基）-3-异喹啉甲酰胺和胆固醇结合。在目前的工作中，我们使用重组PBR和核磁共振的功能和结构特点，这CRAC基序。PBR的C-末端结构域的缺失和CRAC结构域的所有PBR氨基酸序列中高度保守的Y152的突变导致突变体recPBR结合胆固醇的能力丧失。含有CRAC结构域的PBR C-末端肽（144 - 169）的核磁共振分析表明L144 - S159片段具有螺旋构象。作为侧链分布的结果，可以适合胆固醇分子的凹槽一方面由Y152、T148和L144描绘，另一方面由Y153、M149和A145描绘。Y152和Y153的芳环被指定为胆固醇结合的必需残基，构成了凹槽的门。此外，R156的侧链可以通过与甾醇羟基相互作用而覆盖凹槽。这些结果提供了结构和功能的证据支持的发现，CRAC结构域在胞质羧基端结构域的PBR可能是负责的胆固醇的摄取和转运到线粒体。
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-methylpropyl)-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.