Structural interrogation of allosteric AMPK regulation

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消耗能量的细胞过程。amp活化激酶（AMPK）感知人体细胞的能量状态。它被AMP或ADP直接结合激活，并被ATP抑制，两者都竞争AMP/ADP的结合。此外，AMPK受到葡萄糖储存化合物糖原的抑制，糖原与AMPK的一个单独部分结合。活化的AMPK开启atp生成途径，如葡萄糖和脂肪酸的摄取和分解代谢。它还会抑制能量消耗途径，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核心作用，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，需要高分辨率的AMPK在三种相关调节状态（与AMP结合、与AMP和糖原结合、与ATP结合）下的晶体结构，以比较AMP和ATP结合状态以及糖原结合和游离状态。获得这些结构受到使AMPK难以结晶的非结构化区域的阻碍。我们提出了一种方法，经过初步晶体和低分辨率结构的验证，可以修改AMPK的晶体填充表面，使AMPK在其非结构化内部区域和与其变构调节性复合物存在的情况下结晶。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。我们将通过动态氢/氘交换质谱（HDX）以及突变、生化和基于细胞的分析，对AMP和环糊精诱导的表面可达性局部变化进行广泛的定位，从而验证我们的静态晶体结构分析。总之，这些研究的结果将提供AMPK激酶活性变构调节的详细机制，并为合理设计用于治疗糖尿病、肥胖和癌症的新型AMPK调节剂提供结构基础。

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.