Manthei Kelly A, Ahn Joomi, Glukhova Alisa, Yuan Wenmin, Larkin Christopher, Manett Taylor D, Chang Louise, Shayman James A, Axley Milton J, Schwendeman Anna, Tesmer John J G
Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
MedImmune, Gaithersburg, Maryland 20878.
J Biol Chem. 2017 Dec 8;292(49):20313-20327. doi: 10.1074/jbc.M117.802736. Epub 2017 Oct 13.
Lecithin:cholesterol acyltransferase (LCAT) plays a key role in reverse cholesterol transport by transferring an acyl group from phosphatidylcholine to cholesterol, promoting the maturation of high-density lipoproteins (HDL) from discoidal to spherical particles. LCAT is activated through an unknown mechanism by apolipoprotein A-I (apoA-I) and other mimetic peptides that form a belt around HDL. Here, we report the crystal structure of LCAT with an extended lid that blocks access to the active site, consistent with an inactive conformation. Residues Thr-123 and Phe-382 in the catalytic domain form a latch-like interaction with hydrophobic residues in the lid. Because these residues are mutated in genetic disease, lid displacement was hypothesized to be an important feature of apoA-I activation. Functional studies of site-directed mutants revealed that loss of latch interactions or the entire lid enhanced activity against soluble ester substrates, and hydrogen-deuterium exchange (HDX) mass spectrometry revealed that the LCAT lid is extremely dynamic in solution. Upon addition of a covalent inhibitor that mimics one of the reaction intermediates, there is an overall decrease in HDX in the lid and adjacent regions of the protein, consistent with ordering. These data suggest a model wherein the active site of LCAT is shielded from soluble substrates by a dynamic lid until it interacts with HDL to allow transesterification to proceed.
卵磷脂胆固醇酰基转移酶（LCAT）在胆固醇逆向转运中起关键作用，它将酰基从磷脂酰胆碱转移至胆固醇，促使高密度脂蛋白（HDL）从盘状颗粒成熟为球状颗粒。LCAT通过载脂蛋白A-I（apoA-I）和其他在HDL周围形成带状结构的模拟肽以未知机制被激活。在此，我们报道了LCAT的晶体结构，其具有一个延伸的盖子，该盖子阻断了对活性位点的访问，这与非活性构象一致。催化结构域中的苏氨酸-123（Thr-123）和苯丙氨酸-382（Phe-382）残基与盖子中的疏水残基形成类似闩锁的相互作用。由于这些残基在遗传疾病中发生突变，因此推测盖子的移位是apoA-I激活的一个重要特征。定点突变体的功能研究表明，闩锁相互作用的丧失或整个盖子的缺失增强了对可溶性酯底物的活性，氢-氘交换（HDX）质谱分析表明LCAT盖子在溶液中极具动态性。加入一种模拟反应中间体之一的共价抑制剂后，盖子及蛋白质相邻区域的HDX总体下降，这与有序化一致。这些数据提示了一种模型，即LCAT的活性位点被一个动态盖子屏蔽以免接触可溶性底物，直到它与HDL相互作用以使酯交换反应得以进行。
