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）：细胞使用ATP作为“能量货币”，通过将其与ATP水解为ADP/AMP和磷酸盐连接来驱动能量消耗细胞过程。AMP激活的激酶（AMPK）感知人体细胞的能量状态。它被AMP或ADP的直接结合激活，并被ATP抑制，这两者都竞争AMP/ADP结合。此外，AMPK被葡萄糖储存化合物糖原抑制，糖原结合AMPK的单独部分。激活的AMPK开启ATP生成途径，如葡萄糖和脂肪酸的摄取和催化。它还降低了能量消耗途径，如糖原，脂肪酸，胆固醇，rRNA和蛋白质的合成，以及细胞生长和增殖。由于其在葡萄糖代谢和增殖中的中心作用，AMPK是治疗2型糖尿病和癌症的重要治疗靶点。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激酶活性，但这种直接的变构调节机制尚不清楚。理解AMP和糖原如何直接激活和抑制AMPK需要AMPK在三种相关调节状态（与AMP结合、与AMP和糖原结合以及与ATP结合）下的高分辨率晶体结构，以比较AMP和ATP结合状态以及糖原结合和游离状态。获得这些结构受到非结构化区域的阻碍，非结构化区域使AMPK难以结晶。我们提出了一种方法，初步晶体和低分辨率结构验证，修改AMPK的晶体包装表面，允许结晶AMPK在其非结构化的内部区域的存在下，并与其变构调节剂的复合物。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/氘交换质谱（HDX）和突变，生物化学和基于细胞的分析，验证我们的静态晶体结构与广泛的映射o AMP和环糊精诱导的局部变化的表面可及性。总之，所提出的研究的结果将提供AMPK激酶活性的变构调节的详细机制和允许合理设计用于治疗糖尿病、肥胖和癌症的新型AMPK调节剂的结构基础。