Structure, Function and Conformational Dynamics of the Ste14p Methyltransferase

描述（由申请人提供）：本提案侧重于对酿酒酵母Ste14p异戊酰半胱氨酸羧基甲基转移酶（Icmt）的结构、功能和动力学进行深入分析，该酶是一种完整的膜酶，负责大量以c端CaaX基序终止的细胞蛋白的翻译后α-羧基甲基酯化。CaaX蛋白经过三个顺序的翻译后修饰：半胱氨酸残基的异丙烯化，-aaX残基的内蛋白水解，以及Icmt对异丙烯化半胱氨酸的甲基化。甲基化对于CaaX蛋白K-Ras的正确定位至关重要，并且可能是致癌转化的必要条件；因此，Icmt可能被证明是一个很好的化疗靶点。迄今为止，关于Ste14p的分子机制知之甚少，除了它必须容纳化学上不同的甲基供体和受体分子：亲水性辅助因子S-腺苷蛋氨酸（SAM）和亲脂性异戊烯酰化蛋白底物。我们的长期目标是了解这种酶如何在膜/细胞质界面上介导催化的机制细节。具体来说，我们目前的目标是了解Icmt酶识别辅因子和底物的结构-功能关系，并阐明该酶的辅因子结合/催化环的结构动力学。由于哺乳动物Icmts已被证明难以进行功能纯化，我们将使用Ste14p作为我们的模型酶。我们在酵母中过度表达、纯化和功能重组了Ste14p的野生型和无半胱氨酸的变体。我们还在His-Ste14p的保守残基中生成并鉴定了一个大型的位点定向突变文库。使用这些工具，我们现在准备确定构成Ste14p中异戊二烯化底物结合位点的关键残基。我们还建立了识别负责结合辅助因子SAM的重要残基，并揭示SDSL EPR结合辅助因子和/或脂化底物时酶中发生的构象变化，以基于细菌Icmt同源物晶体结构的Ste14p c端催化半部分的结构同源模型为指导。总之，这些研究将为Ste14p识别底物和辅因子的本质提供关键见解，从而更好地理解其作用机制。考虑到Ste14p与其他Icmt在结构、功能和序列上的相似性，对酵母酶的研究将为进一步了解这类酶的其他成员的功能提供一般的机制，最终可能为合理设计针对人类Icmt的抗癌药物提供基础。

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.