Structure, Function and Conformational Dynamics of the Ste14p Methyltransferase

Ste14p 甲基转移酶的结构、功能和构象动力学

基本信息

  • 批准号:
    9040994
  • 负责人:
  • 金额:
    $ 27.77万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2013
  • 资助国家:
    美国
  • 起止时间:
    2013-07-01 至 2018-03-31
  • 项目状态:
    已结题

项目摘要

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.
描述(由申请人提供):这项建议集中在深入分析来自酿酒酵母的Ste14p异丙基半胱氨酸羧甲基转移酶(ICMT)的结构、功能和动力学,该酶是负责大量细胞蛋白翻译后α-羧甲基甲酯化的完整膜酶,这些蛋白质终止于C-末端CAAX基序。CAAX蛋白经历了三个顺序的翻译后修饰:半胱氨酸残基的异戊二烯基化,-AAX残基的内切蛋白降解,以及ICMT对异丙二烯基化的半胱氨酸的甲基化。甲基化对于CAAX蛋白K-RAS的正确定位是至关重要的,也可能是致癌转化所必需的;因此,ICMT可能被证明是一个很好的化疗靶点。到目前为止,人们对Ste14p的作用机制知之甚少,除了它必须适应化学上不同的甲基供体和受体分子:亲水性辅助因子S-腺苷蛋氨酸(SAM)和亲脂性异戊二烯基化蛋白底物。我们的长期目标是从机制上详细了解这种酶是如何在膜/胞浆界面上介导催化作用的。具体地说,我们在本提案中的目标是了解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,因此我们将使用Ste14p作为我们的模型酶。我们已经在酵母中高效表达、纯化和功能重组了毫克量的野生型和无半胱氨酸变异体Ste14p。我们还在His-Ste14p的保守残基中产生并鉴定了一个大型定点突变体文库。使用这些工具,我们现在可以确定组成ST14P中异戊二烯基底物结合位点的关键残基。我们还建立了识别负责结合辅因子SAM的重要残基,并在SDSL EPR与辅因子和/或脂化底物结合时揭示酶中发生的构象变化,该模型是基于细菌ICMT直系物的晶体结构而建立的。总之,这些研究将为Ste14p识别底物和辅因子的性质提供关键的见解,从而更好地了解其作用机制。鉴于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了解这类酶的其他成员的功能提供一般性的机制洞察,最终可能为针对人ICMT的抗癌药物的合理设计提供基础。

项目成果

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CHRISTINE A HRYCYNA其他文献

CHRISTINE A HRYCYNA的其他文献

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{{ truncateString('CHRISTINE A HRYCYNA', 18)}}的其他基金

FASEB SRC on Protein Lipidation, Signaling and Membrane Domains.
FASEB SRC 关于蛋白质脂化、信号传导和膜结构域。
  • 批准号:
    8977960
  • 财政年份:
    2015
  • 资助金额:
    $ 27.77万
  • 项目类别:
Structure, Function and Conformational Dynamics of the Ste14p Methyltransferase
Ste14p 甲基转移酶的结构、功能和构象动力学
  • 批准号:
    8483309
  • 财政年份:
    2013
  • 资助金额:
    $ 27.77万
  • 项目类别:
Structure, Function and Conformational Dynamics of the Ste14p Methyltransferase
Ste14p 甲基转移酶的结构、功能和构象动力学
  • 批准号:
    8675864
  • 财政年份:
    2013
  • 资助金额:
    $ 27.77万
  • 项目类别:
FASEB SRC on Protein Lipidation, Signaling and Membrane Domains
FASEB SRC 关于蛋白质脂化、信号传导和膜结构域
  • 批准号:
    8203842
  • 财政年份:
    2011
  • 资助金额:
    $ 27.77万
  • 项目类别:
Modulating P-glycoprotein to Enhance Neurodegenerative Drug Penetration of Brain
调节 P-糖蛋白增强神经退行性药物在大脑中的渗透
  • 批准号:
    7489886
  • 财政年份:
    2007
  • 资助金额:
    $ 27.77万
  • 项目类别:
Modulating P-glycoprotein to Enhance Neurodegenerative Drug Penetration of Brain
调节 P-糖蛋白增强神经退行性药物在大脑中的渗透
  • 批准号:
    7329774
  • 财政年份:
    2007
  • 资助金额:
    $ 27.77万
  • 项目类别:

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