Structural interrogation of allosteric AMPK regulation

变构 AMPK 调节的结构探究

• 批准号: 8584776
• 负责人: Karsten Melcher
• 金额: $ 38.32万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8584776
• 关键词: 5' -AMP-activated protein kinase; ATP Hydrolysis; Aging; Allosteric Regulation; Autophagocytosis; Binding; Biochemical; Biological Assay; Carbohydrates; Catabolism; Catalytic Domain; Cell Proliferation; Cell physiology; Cells; Cholesterol; Complex; Crystallization; Cyclodextrins; Data; Deuterium; Diabetes Mellitus; Dissociation; Drops; Fatty Acids; Glucose; Glycogen; Growth; Homeostasis; Human; Hydrogen; Length; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Metabolic Diseases; Metabolism; Mitochondria; Molecular Conformation; Mutate; Non-Insulin-Dependent Diabetes Mellitus; Nucleotides; Obesity; Outcome; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Proteins; Regulation; Resolution; Ribosomal RNA; Role; Stress; Structure; Surface; base; cancer therapy; cell growth; design; glucose metabolism; inorganic phosphate; luminescence; neoplastic cell; novel; novel therapeutics; programs; public health relevance; sensor; therapeutic target; upstream kinase; uptake

DESCRIPTION (provided by applicant): Cells use ATP as "energy currency" to drive energy consuming cellular processes by linking them to the hydrolysis of ATP to ADP/AMP and phosphate. AMP-activated kinase (AMPK) senses the energy status in human cells. It becomes activated by the direct binding of AMP or ADP and is inhibited by ATP, which both compete for AMP/ADP binding. In addition, AMPK is inhibited by the glucose storage compound glycogen, which binds to a separate part of AMPK. Activated AMPK turns on ATP-generating pathways, such as glucose and fatty acid uptake and catabolism. It also turns down energy-consuming pathways, such as the synthesis of glycogen, fatty acids, cholesterol, rRNA, and proteins, as well as cell growth and proliferation. Due to its central roles in glucose metabolism and proliferation, AMPK is an important therapeutic target for the treatment of type 2 diabetes and cancer. AMP and ADP activate AMPK by changing AMPK's accessibility to upstream regulators (kinases and phosphatases). In addition, AMP and glycogen directly activate and inhibit the AMPK kinase activity, respectively, but the mechanism of this direct, allosteric regulation is unknown. Understanding how AMP and glycogen directly activate and inhibit AMPK requires high resolution crystal structures of AMPK in three relevant regulatory states (bound to AMP, bound to AMP and glycogen, and bound to ATP) to compare AMP- and ATP-bound states as well glycogen-bound and -free states. Obtaining these structures is hampered by unstructured regions that make AMPK recalcitrant to crystallization. We present an approach, validated by preliminary crystals and low resolution structures, to modify AMPK's crystal packing surfaces to allow the crystallization of AMPK in the presence of its unstructured internal regions and in complexes with its allosteric modulators. Comparison of these structures will allow us to identify changes in interactions and conformations associated with allosteric AMPK activation and inhibition. We will validate the analysis of our static crystal structures with extensive mapping o AMP- and cyclodextrin-induced local changes in surface accessibility by dynamic hydrogen/deuterium exchange mass spectrometry (HDX) and by mutational, biochemical, and cell-based analyses. Together, the results of the proposed studies will provide a detailed mechanism of the allosteric regulation of AMPK kinase activity and a structural basis to allow the rational design of novel AMPK modulators for the treatment of diabetes, obesity, and cancer.

描述（由申请人提供）：细胞将ATP用作“能量货币”，通过将它们与ATP的水解与ADP/AMP和磷酸盐的水解联系起来来驱动消耗细胞过程。 AMP激活的激酶（AMPK）感觉到人类细胞中的能量状态。它被AMP或ADP的直接结合激活，并被ATP抑制，ATP都竞争AMP/ADP结合。另外，AMPK受到葡萄糖储存化合物糖原的抑制，该糖原与AMPK的单独部分结合。激活的AMPK打开了ATP生成的途径，例如葡萄糖和脂肪酸摄取和分解代谢。它还降低了消耗的途径，例如糖原，脂肪酸，胆固醇，rRNA和蛋白质的合成，以及细胞的生长和增殖。由于其在葡萄糖代谢和增殖中的主要作用，AMPK是治疗2型糖尿病和癌症的重要治疗靶标。 AMP和ADP通过更改AMPK对上游调节剂（激酶和磷酸酶）的可访问性来激活AMPK。另外，AMP和糖原分别直接激活和抑制AMPK激酶活性，但是这种直接的，变构调节的机制尚不清楚。了解AMP和糖原如何直接激活和抑制AMPK需要在三个相关调节状态（与AMP结合，与AMP和糖原结合，与ATP结合，并与ATP结合）的AMPK高分辨率晶体结构，以比较AMP和ATP结合状态以及Glycogen - 绑定和 - 无状态。获得这些结构的非结构化区域会阻碍，使AMPK顽固地升级为结晶。我们提出了一种通过初步晶体和低分辨率结构验证的方法，以修改AMPK的晶体堆积表面，以使AMPK在其非结构化内部区域的存在下以及具有变构调节剂的复合物中的结晶。这些结构的比较将使我们能够确定与变构AMPK激活和抑制相关的相互作用和构象的变化。我们将通过大量映射OAMP和环糊精诱导的局部晶体结构进行验证分析，并通过动态氢/氘交换质谱（HDX）以及突变，生化和基于细胞的分析，从而通过动态氢/氘交换质谱法（HDX）进行了局部变化。共同的研究结果将提供AMPK激酶活性的变构调节和结构性基础的详细机制，以允许新型AMPK调节剂的理性设计用于治疗糖尿病，肥胖和癌症。