Structure and mechanism of the AMP-activated protein kinase

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水平的变化来协调真核生物中的代谢与能量可用性。AMPK的激酶活性被AMP刺激并被过量的ATP抑制，并且认为这种独特的调节行为使AMPK能够充当中央细胞“燃料计”。因此，AMPK作为治疗代谢紊乱如糖尿病和肥胖症的治疗剂的靶标是非常感兴趣的对象。AMPK是abg异源三聚体，其包括具有丝氨酸/苏氨酸激酶活性的亚基和由来自所有三个亚基的元件组成的腺苷酸结合调节区。在这个应用程序的初步数据，我们目前的晶体结构AMP和ATP结合形式的异源三聚体腺苷酸传感器裂殖酵母酶。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，这有助于解释它们的竞争效应。生物物理实验表明，腺苷酸传感器复合物结合的α亚基激酶结构域的AMP的存在下，但ATP结合阻止这种协会。这些数据有助于提供AMPK调节的初步分子理解。令人惊讶的是，AMPK-ADP复合物的晶体结构揭示了可以独特地容纳ADP的第二结合位点。本申请的首要目标是通过以下具体目标获得对AMPK调节的原子水平理解：（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来确定配体结合如何影响调节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，我们将使用定点诱变来定义负责激酶结构域和调节腺苷酸传感器之间的核苷酸依赖性关联的分子区域。从拟议的工作结果将是至关重要的AMPK导向疗法的合理发展。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有吸引力的分子靶点之一。先前的研究已经表明，给予糖尿病动物的AMPK激活剂可以显著改善糖尿病的生理效应。尽管AMPK导向疗法前景广阔，但对调控的分子机制知之甚少，并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受到阻碍。我们的初步结果和进一步的工作将提供高分辨率的结构信息AMPK，并应直接使AMPK导向治疗的合理设计。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有吸引力的分子靶点之一。先前的研究已经表明，给予糖尿病动物的AMPK激活剂可以显著改善糖尿病的生理效应。尽管AMPK导向疗法前景广阔，但对调控的分子机制知之甚少，并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受到阻碍。我们的初步结果和进一步的工作将提供高分辨率的结构信息AMPK，并应直接使AMPK导向治疗的合理设计。

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.