Human CaaX protease ZMPSTE24 expressed in yeast: Structure and inhibition by HIV protease inhibitors
Human CaaX protease ZMPSTE24 expressed in yeast: Structure and inhibition by HIV protease inhibitors
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DOI：
10.1002/pro.3074
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发表时间：
2017-02-01
期刊：
影响因子：
8
通讯作者：
Dumont, Mark E.
中科院分区：
文献类型：
--
作者：
Clark, Kathleen M.;Jenkins, Jermaine L.;Dumont, Mark E.
含有c-末端CaaX（半胱氨酸-脂肪-脂肪-任何）序列基序的蛋白质和肽的功能和定位是通过翻译后异戊烯基与半胱氨酸巯基的结合以及aaX氨基酸的蛋白水解裂解来调节的。锌金属蛋白酶ZMPSTE24是已知的催化这种裂解的两种酶之一。哺乳动物ZMPSTE24唯一确定的靶点是核支架蛋白lamin A的前体prelamin A。ZMPSTE24还在CaaX位点上游15个残基的第二个位点切割prelamin A。ZMPSTE24的突变导致过早衰老疾病，ZMPSTE24活性的抑制已被报道为HIV蛋白酶抑制剂的脱靶效应。我们在此报告了人类ZMPSTE24的表达（在酵母中）、纯化和结晶，从而可以在2.0埃分辨率下确定其结构。与之前的低分辨率结构相比，增强的分辨率提供了：(1)ZMPSTE24活性位点的详细视图，包括配位催化锌的水；(2)增强了从蛋白质的外部到内部腔的通道的可视化；(3)从蛋白质主体向外延伸的c端视图；(4)有序的脂质和洗涤剂分子在内外表面的定位，也通过开窗投射；(5)识别与内腔表面相关的水分子。我们还对纯化的ZMPSTE24的活性进行了荧光测定，以证明HIV蛋白酶抑制剂以一种表明竞争机制的方式直接抑制人类酶。PDB代码:
The function and localization of proteins and peptides containing C-terminal CaaX (Cys-aliphatic-aliphatic-anything) sequence motifs are modulated by post-translational attachment of isoprenyl groups to the cysteine sulfhydryl, followed by proteolytic cleavage of the aaX amino acids. The zinc metalloprotease ZMPSTE24 is one of two enzymes known to catalyze this cleavage. The only identified target of mammalian ZMPSTE24 is prelamin A, the precursor to the nuclear scaffold protein lamin A. ZMPSTE24 also cleaves prelamin A at a second site 15 residues upstream from the CaaX site. Mutations in ZMPSTE24 result in premature-aging diseases and inhibition of ZMPSTE24 activity has been reported to be an off-target effect of HIV protease inhibitors. We report here the expression (in yeast), purification, and crystallization of human ZMPSTE24 allowing determination of the structure to 2.0 angstrom resolution. Compared to previous lower resolution structures, the enhanced resolution provides: (1) a detailed view of the active site of ZMPSTE24, including water coordinating the catalytic zinc; (2) enhanced visualization of fenestrations providing access from the exterior to the interior cavity of the protein; (3) a view of the C-terminus extending away from the main body of the protein; (4) localization of ordered lipid and detergent molecules at internal and external surfaces and also projecting through fenestrations; (5) identification of water molecules associated with the surface of the internal cavity. We also used a fluorogenic assay of the activity of purified ZMPSTE24 to demonstrate that HIV protease inhibitors directly inhibit the human enzyme in a manner indicative of a competitive mechanism.PDB Code: