Chemical biology of protein lipidation: semi-synthesis and structure elucidation of prenylated RabGTPases
Chemical biology of protein lipidation: semi-synthesis and structure elucidation of prenylated RabGTPases
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DOI：
10.1039/b417573e
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发表时间：
2005-01-01
影响因子：
3.2
通讯作者：
Waldmann, H
中科院分区：
文献类型：
--
作者：
Watzke, A;Brunsveld, L;Waldmann, H
RAB/YPT鸟苷三磷酸酶(GTP酶)是真核细胞中一类重要的膜交通调节因子。为了发挥其功能，Rab/YPT蛋白需要在C末端用两个共价结合的香叶基脂类部分进行双重修饰。通常情况下，异戊烯基化蛋白质很难通过重组或酶法获得。我们利用化学合成和蛋白质工程相结合的方法产生了预烯基化的RabGTP酶。这种半合成在很大程度上取决于与蛋白质的天然结构或修饰相对应的功能化的戊烯基化肽的可用性。我们开发了溶液相和固相策略来生成与Rab7 GTPase的预烯化C-末端相对应的多肽，使我们能够结晶单戊烯基化的Ypt1：RabGDI复合体。该复合体的结构为RabGDI抑制Rab蛋白释放核苷酸的能力提供了结构基础，也为理解导致人类智力低下的RabGDI突变提供了分子基础。
Rab/Ypt guanosine triphosphatases (GTPases) represent a family of key membrane traffic regulators in eukaryotic cells. For their function Rab/Ypt proteins require double modification with two covalently bound geranylgeranyl lipid moieties at the C-terminus. Generally, prenylated proteins are very difficult to obtain by recombinant or enzymatic methods. We generated prenylated RabGTPases using a combination of chemical synthesis and protein engineering. This semi-synthesis depends largely on the availability of functionalized prenylated peptides corresponding to the proteins' native structure or modifications. We developed solution phase and solid phase strategies for the generation of peptides corresponding to the prenylated C-terminus of Rab7 GTPase in preparative amounts enabling us to crystallize the mono-prenylated Ypt1:RabGDI complex. The structure of the complex provides a structural basis for the ability of RabGDI to inhibit the release of nucleotide by Rab proteins and a molecular basis for understanding a RabGDI mutant that causes mental retardation in humans.