Structure and mechanism of the AMP-activated protein kinase

AMP激活蛋白激酶的结构和机制

• 批准号: 7901042
• 负责人: LAWRENCE S SHAPIRO
• 金额: $ 31.66万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7901042
• 关键词: 5' -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

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活化蛋白激酶（AMPK）通过响应细胞内ATP和AMP水平的变化，协调真核生物的代谢和能量可用性。AMPK的激酶活性受到AMP的刺激，并受到过量ATP的抑制，人们认为这种独特的调节行为使AMPK能够作为中心细胞的“燃料计”。因此，AMPK作为治疗代谢紊乱（如糖尿病和肥胖症）的靶点而备受关注。AMPK是一种abg异源三聚体，包括一个具有丝氨酸/苏氨酸激酶活性的亚基和一个由所有三个亚基组成的腺苷酸结合调节区。在此应用的初步数据中，我们展示了来自Schizosacharomyces pombe酶的AMP和atp结合形式的异三聚体腺苷酸传感器的晶体结构。该复合物缺乏激酶催化结构域，但揭示了ampk的保守三聚体核心结构。ATP和AMP竞争性地结合到g亚基内的单个位点上，这有助于解释它们的竞争性作用。生物物理实验表明腺苷酸传感器复合物在AMP存在的情况下结合a亚基激酶结构域，但ATP的结合阻止了这种结合。这些数据有助于提供对AMPK调控的初步分子理解。令人惊讶的是，AMPK-ADP复合物的晶体结构揭示了第二个结合位点，可以独特地容纳ADP。本应用程序的总体目标是通过以下具体目标获得对AMPK调控的原子水平的理解：(1)表征各种腺苷酸配体结合的亲和力，并使用生物物理方法确定配体结合如何影响调节结构域和激酶结构域之间的相互作用。这些研究的结果将与各种配体结合状态下的激酶活性相关。(2)为了了解全酶的结构，我们将使用位点定向诱变来定义激酶结构域和调节腺苷酸传感器之间的核苷酸依赖关联的分子区域。这项工作的结果将对ampk导向疗法的合理开发至关重要。AMPK是细胞代谢的中心调节因子，是治疗糖尿病、肥胖和其他代谢紊乱的新疗法中最具吸引力的分子靶点之一。先前的研究表明，AMPK激活剂给予糖尿病动物可以显著改善糖尿病的生理效应。尽管以ampk为导向的治疗方法前景广阔，但人们对其调控的分子机制知之甚少，而且由于缺乏这种酶结构的原子水平信息，设计合适的小分子药物受到了阻碍。我们的初步结果和进一步的工作将提供关于AMPK的高分辨率结构信息，并将直接使AMPK导向治疗的合理设计成为可能。公共卫生相关性：AMPK是细胞代谢的中心调节因子，是治疗糖尿病、肥胖和其他代谢紊乱的新疗法中最具吸引力的分子靶点之一。先前的研究表明，AMPK激活剂给予糖尿病动物可以显著改善糖尿病的生理效应。尽管以ampk为导向的治疗方法前景广阔，但人们对其调控的分子机制知之甚少，而且由于缺乏这种酶结构的原子水平信息，设计合适的小分子药物受到了阻碍。我们的初步结果和进一步的工作将提供关于AMPK的高分辨率结构信息，并将直接使AMPK导向治疗的合理设计成为可能。