Structure and mechanism of the AMP-activated protein kinase

说明(申请人提供)：AMPK通过反应细胞内ATP和AMP水平的变化来协调真核生物的新陈代谢和能量供应。AMPK的激酶活性受到AMP的刺激，而被过量的ATP抑制，这一独特的调节行为被认为使AMPK成为细胞的中枢性“油量表”。因此，AMPK是治疗糖尿病和肥胖症等代谢性疾病的治疗靶点，引起了人们的强烈兴趣。AMPK是一种ABG异源三聚体，它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的元件组成的腺苷结合调节区。在这一应用的初步数据中，我们介绍了来自裂殖酵母庞贝裂殖酵母酶的异三聚腺苷传感器的AMP和ATP结合形式的晶体结构。该复合体缺乏激酶催化结构域，但揭示了AMPKs保守的三聚体核心结构。ATP和AMP竞争性地结合到g亚基内的单个位点，有助于解释它们的竞争效应。生物物理实验表明，腺苷类传感器复合体在AMP存在的情况下与α亚单位激酶结构域结合，但ATP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复合体的晶体结构揭示了第二个能够唯一容纳ADP的结合位点。本申请的首要目标是通过以下具体目标在原子水平上了解AMPK的调控：(1)表征各种腺苷配体结合的亲和力，并使用生物物理方法确定配体结合如何影响调节域和激活域之间的相互作用。这些研究的结果将与不同配体结合状态下的激酶活性相关联。(2)为了了解全酶的结构，我们将使用定点突变来定义负责激酶域和调节腺苷酸传感器之间核苷酸依赖性结合的分子区域。拟议工作的结果将是AMPK导向疗法的合理发展的关键。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最具吸引力的分子靶点之一。先前的研究表明，AMPK激活剂应用于糖尿病动物可以显著改善糖尿病的生理效应。尽管AMPK指导的疗法前景光明，但人们对其调控的分子机制知之甚少，而且由于缺乏关于酶结构的原子水平信息，适当的小分子药物的设计一直受到阻碍。我们的初步结果和下一步工作将提供AMPK的高分辨率结构信息，并将直接使AMPK导向疗法的合理设计成为可能。公共卫生相关性：AMPK是一种细胞代谢的中央调节剂，是治疗糖尿病、肥胖症和其他代谢紊乱的新疗法最具吸引力的分子靶点之一。先前的研究表明，AMPK激活剂应用于糖尿病动物可以显著改善糖尿病的生理效应。尽管AMPK指导的疗法前景光明，但人们对其调控的分子机制知之甚少，而且由于缺乏关于酶结构的原子水平信息，适当的小分子药物的设计一直受到阻碍。我们的初步结果和下一步工作将提供AMPK的高分辨率结构信息，并将直接使AMPK导向疗法的合理设计成为可能。

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.