Structure, Function and Conformational Dynamics of the Ste14p Methyltransferase

描述（由申请人提供）：本提案侧重于深入分析来自S.酿酒酵母，一种整合的膜酶，负责终止于C-末端CaaX基序的大量细胞蛋白的翻译后α-羧甲基酯化。CaaX蛋白经历三个连续的翻译后修饰：异戊二烯化的半胱氨酸残基，内切蛋白水解的-aaX残基，和甲基化的异戊二烯化的半胱氨酸的ICMT。甲基化对于CaaX蛋白K-Ras的适当定位是至关重要的，并且对于致癌转化可能是必不可少的;因此，Icmt可能被证明是极好的化疗靶点。 到目前为止，很少有分子的细节是已知的机制Ste 14 p，除了它必须适应化学上不同的甲基供体和受体分子：亲水性辅因子S-腺苷甲硫氨酸（SAM）和亲脂性异戊二烯化蛋白质底物，分别。我们的长期目标是详细了解这种酶如何在膜/胞质界面介导催化作用。具体而言，我们的目标是在目前的建议是了解辅因子和底物识别的ICMT酶的结构-功能关系，并阐明辅因子结合/催化环的酶的结构动力学。由于哺乳动物ICMTS已被证明难以进行功能纯化，我们将使用Ste 14 p作为我们的模型酶。 我们已经过表达，纯化和功能重建毫克量的野生型和半胱氨酸的变体Ste 14 p在酵母中。我们还产生并表征了His-Ste 14 p保守残基中的定点突变体的大文库。使用这些工具，我们现在准备确定关键的残基，包括在Ste 14 p的异戊二烯化底物的结合位点。我们还建立了确定重要的残基负责的辅因子SAM的结合，并揭示构象变化时，发生在酶的辅因子和/或脂化底物结合SDSL EPR，由结构同源性模型的C-末端催化的一半的Ste 14 p是基于细菌Icmt直系同源物的晶体结构的指导。 总之，这些研究将提供关键的洞察底物和辅因子识别的性质，通过STE 14 p，将使其作用机制有一个更好的理解。考虑到Ste 14 p和其他Icmt之间的结构，功能和序列相似性，酵母酶的研究应该提供一般的机制洞察到这类酶的其他成员的功能，最终可能提供的基础上，合理设计的抗癌药物靶向人类Icmt。

DESCRIPTION (provided by applicant): This proposal focuses on an in-depth analysis of the structure, function and dynamics of the Ste14p isoprenylcysteine carboxyl methyltransferase (Icmt) from S. cerevisiae, the integral membrane enzyme that is responsible for the posttranslational α-carboxyl methyl esterification of a large number of cellular proteins that terminate in a C-terminal CaaX motif. CaaX proteins undergo three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the -aaX residues, and methylation of the isoprenylated cysteine by Icmt. Methylation is critical for the proper localization of the CaaX protein K-Ras and may be essential for oncogenic transformation; thus, Icmt may prove to be an excellent chemotherapeutic target. To date, few molecular details are known about the mechanism of Ste14p except that it must accommodate chemically diverse methyl donor and acceptor molecules: the hydrophilic co-factor S- adenosylmethionine (SAM) and a lipophilic isoprenylated protein substrate, respectively. Our long term goal is to understand in mechanistic detail how such an enzyme mediates catalysis at the membrane/cytosol interface. Specifically, our goals in the current proposal are to understand the structure-function relationships of co-factor and substrate recognition by the Icmt enzyme and to elucidate the structural dynamics of the co- factor binding/catalytic loop of the enzyme. As mammalian Icmts have proven intractable to functional purification, we will use Ste14p, as our model enzyme. We have overexpressed, purified and functionally reconstituted milligram quantities of both wild-type and a cysteine-less variant of Ste14p in yeast. We have also generated and characterized a large library of site- directed mutants in conserved residues of His-Ste14p. Using these tools, we are now poised to identify key residues that comprise the binding sites in Ste14p for isoprenylated substrates. We are also set up to identify important residues responsible for binding of the co-factor SAM and to reveal conformational changes that occur in the enzyme upon co-factor and/or lipidated substrate binding by SDSL EPR, guided by a structural homology model of the C-terminal catalytic half of Ste14p that is based on the crystal structure of a bacterial Icmt ortholog. Together, these studies will provide key insights into the nature of substrate and co-factor recognition by Ste14p that will give a better understanding of its mechanism of action. Given the structural, functional and sequence similarities between Ste14p and other Icmts, study of the yeast enzyme should offer general mechanistic insight into the function of other members of this class of enzymes which ultimately may provide the basis for the rational design of anti-cancer drugs targeting human Icmt.