Structure and mechanism of the AMP-activated protein kinase

AMP激活蛋白激酶的结构和机制

基本信息

批准号：
7523548
负责人：
LAWRENCE S SHAPIRO
金额：
$ 31.54万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7523548
关键词：
5&apos -AMP-activated protein kinase Adipocytes Affect Affinity Animals Architecture Behavior Binding Binding Sites Biological Assay Catalytic Domain Complex Data Development Diabetes Mellitus Elements Enzymes Eukaryota Evaluation Exercise Fission Yeast Glucose Goals Holoenzymes Human Ligand Binding Ligands Light Maps Mass Spectrum Analysis Measures Metabolic Diseases Metabolism Methods Molecular Molecular Target Muscle Fibers Mutagenesis Nucleic Acid Regulatory Sequences Nucleotides Obesity Organism Pharmaceutical Preparations Phosphotransferases Physiological Protein-Serine-Threonine Kinases Proteolysis Regulation Regulatory Element Resolution Site Site-Directed Mutagenesis Structure Therapeutic Work adenylate base design diabetic enzyme structure interest novel therapeutics prevent public health relevance research study response sensor small molecule therapeutic target

项目摘要

DESCRIPTION (provided by applicant): AMP-activated protein kinase (AMPK) coordinates metabolism with energy availability in eukaryotes by responding to changes in intracellular ATP and AMP levels. The kinase activity of AMPK is stimulated by AMP and inhibited by excess ATP, and it is thought that this unique regulatory behavior enables AMPK to act as a central cellular "fuel gauge". AMPK is thus the subject of intense interest as a target for therapeutics to treat metabolic disorders such as diabetes and obesity. AMPK is an abg heterotrimer that includes a subunit with serine/threonine kinase activity and an adenylate-binding regulatory region composed of elements from all three subunits. In preliminary data for this application we present crystal structures for AMP- and ATP-bound forms of the heterotrimeric adenylate sensor from the Schizosacharomyces pombe enzyme. This complex lacks the kinase catalytic domain, but reveals the conserved trimeric core architecture of AMPKs. ATP and AMP bind competitively to a single site within the g subunit, helping to explain their competing effects. Biophysical experiments show that the adenylate sensor complex binds the a subunit kinase domain in the presence of AMP but ATP binding prevents this association. These data help to provide an initial molecular understanding of AMPK regulation. A crystal structure of an AMPK-ADP complex, surprisingly, reveals a second binding site that can uniquely accommodate ADP. The overarching goal of this application is to gain an atomic-level understanding of AMPK regulation through the following specific aims: (1) characterizes the affinities of binding of various adenylate ligands, and use biophysical methods to determine how ligand binding affects interaction between the regulatory and kinase domains. Results from these studies will be correlated with kinase activity in various ligand-bound states. (2) To gain an understanding of the holoenzyme architecture, we will use site-directed mutagenesis to define the molecular regions responsible for nucleotide-dependent association between the kinase domain and regulatory adenylate sensor. Results from the proposed work will be critical for the rational development of AMPK-directed therapeutics. AMPK, a central regulator of cellular metabolism, is among the most attractive molecular targets for new therapeutics to treat diabetes, obesity, and other metabolic disorders. Prior studies have shown that activators of AMPK administered to diabetic animals can substantially ameliorate the physiological effects of diabetes. Despite the great promise of AMPK-directed therapeutics, little is known about the molecular mechanisms of regulation, and the design of appropriate small molecule drugs has been impeded by the lack of atomic-level information on the architecture of the enzyme. Our preliminary results and the further work proposed will provide high-resolution structural information on AMPK, and should directly enable the rational design of AMPK-directed therapeutics. PUBLIC HEALTH RELEVANCE: AMPK, a central regulator of cellular metabolism, is among the most attractive molecular targets for new therapeutics to treat diabetes, obesity, and other metabolic disorders. Prior studies have shown that activators of AMPK administered to diabetic animals can substantially ameliorate the physiological effects of diabetes. Despite the great promise of AMPK-directed therapeutics, little is known about the molecular mechanisms of regulation, and the design of appropriate small molecule drugs has been impeded by the lack of atomic-level information on the architecture of the enzyme. Our preliminary results and the further work proposed will provide high-resolution structural information on AMPK, and should directly enable the rational design of AMPK-directed therapeutics.

描述（由申请人提供）：AMP激活的蛋白激酶（AMPK）通过响应细胞内ATP和AMP水平的变化，使真核生物的能量可用性与真核生物的能量可用。 AMPK的激酶活性被AMP刺激并被过量的ATP抑制，并且人们认为这种独特的调节行为使AMPK能够充当中央细胞“燃油表”。因此，AMPK是强烈兴趣的主题，是治疗糖尿病和肥胖症等代谢疾病的治疗剂的靶标。 AMPK是一种ABG异三聚体，其中包括具有丝氨酸/苏氨酸激酶活性的亚基和一个由所有三个亚基的元素组成的腺苷酸盐结合调节区域。在此应用的初步数据中，我们提出了来自schizosacharomyces pombe酶的杂点腺苷传感器的AMP和ATP结合形式的晶体结构。该复合物缺乏激酶催化域，但揭示了AMPK的保守三聚体核心结构。 ATP和AMP竞争与G亚基内的单个站点结合，有助于解释其竞争效果。生物物理实验表明，在存在AMP的情况下，腺苷酸传感器复合物结合了A亚基激酶结构域，但ATP结合阻止了这种关联。这些数据有助于提供对AMPK调节的初始分子理解。令人惊讶的是，AMPK-ADP复合物的晶体结构揭示了一个可以独特容纳ADP的第二个结合位点。该应用程序的总体目标是通过以下特定目的获得对AMPK调节的原子水平的理解：（1）表征各种腺苷酸配体的结合亲和力，并使用生物物理方法来确定配体结合如何影响调节性和激酶域之间的相互作用。这些研究的结果将与各种配体结合状态的激酶活性相关。（2）为了了解全酶体系结构，我们将使用位置定向的诱变来定义负责激酶结构域与调节腺苷酸腺苷酸酯传感器之间核苷酸依赖性关联的分子区域。拟议工作的结果对于AMPK定向治疗剂的合理发展至关重要。 AMPK是细胞代谢的中心调节剂，是治疗糖尿病，肥胖症和其他代谢疾病的新疗法的最具吸引力的分子靶标之一。先前的研究表明，施用对糖尿病动物的AMPK的激活剂可以显着改善糖尿病的生理作用。尽管有AMPK指导的治疗剂的巨大希望，但对调节的分子机制知之甚少，并且由于缺乏有关酶体系结构的原子级信息而阻碍了适当的小分子药物的设计。我们的初步结果以及提出的进一步工作将提供有关AMPK的高分辨率结构信息，并应直接实现AMPK指导治疗剂的合理设计。公共卫生相关性：AMPK是细胞代谢的中心调节剂，是治疗糖尿病，肥胖和其他代谢疾病的新疗法的最有吸引力的分子靶标之一。先前的研究表明，施用对糖尿病动物的AMPK的激活剂可以显着改善糖尿病的生理作用。尽管有AMPK指导的治疗剂的巨大希望，但对调节的分子机制知之甚少，并且由于缺乏有关酶体系结构的原子级信息而阻碍了适当的小分子药物的设计。我们的初步结果以及提出的进一步工作将提供有关AMPK的高分辨率结构信息，并应直接实现AMPK指导治疗剂的合理设计。