Structural interrogation of allosteric AMPK regulation

变构 AMPK 调节的结构探究

• 批准号: 8831699
• 负责人: Karsten Melcher
• 金额: $ 36.22万
• 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 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8831699
• 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; cell growth; design; glucose metabolism; inorganic phosphate; luminescence; neoplastic cell; novel; novel therapeutics; programs; public health relevance; sensor; targeted cancer therapy; 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(申请人提供)：细胞使用三磷酸腺苷作为“能量货币”，通过将三磷酸腺苷与二磷酸腺苷/二磷酸腺苷和磷酸盐的水解酶联系起来，来驱动消耗能量的细胞过程。AMPK(AMPK)是一种感知人体细胞内能量状态的信号通路。它被AMP或ADP的直接结合激活，并被竞争AMP/ADP结合的ATP抑制。此外，AMPK被葡萄糖储存化合物糖原抑制，该糖原与AMPK的单独部分结合。激活的AMPK启动了ATP的生成途径，如葡萄糖和脂肪酸的吸收和分解代谢。它还可以抑制消耗能量的途径，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AMPK在葡萄糖代谢和增殖中的核心作用，它是治疗2型糖尿病和癌症的重要靶点。AMP和ADP通过改变AMPK对上游调节因子(激酶和磷酸酶)的可及性来激活AMPK。此外，AMP和糖原分别直接激活和抑制AMPK活性，但这种直接的变构调节机制尚不清楚。要了解AMP和糖原是如何直接激活和抑制AMPK的，需要AMPK在三个相关调控状态(结合AMP、结合AMP和糖原、结合ATP)下的高分辨率晶体结构来比较AMP和ATP结合状态以及糖原结合和游离状态。这些结构的获得受到非结构化区域的阻碍，这些区域使AMPK抵抗结晶。我们提出了一种方法，通过初步晶体和低分辨结构的验证，来修饰AMPK的晶体堆积表面，以允许AMPK在其非结构内部区域存在的情况下和在其变构调节剂的络合物中结晶。对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。我们将通过动态氢/氢交换质谱仪(HDX)以及突变、生化和基于细胞的分析，通过对AMP和环糊精引起的表面可及性局部变化的广泛映射来验证我们的静态晶体结构分析。综上所述，这些研究结果将提供变构调节AMPK活性的详细机制，并为合理设计用于治疗糖尿病、肥胖症和癌症的新型AMPK调节剂提供结构基础。