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.

Public Health Relevance

AMP-activated kinase (AMPK) is an important therapeutic target for the treatment of type 2 diabetes, obesity, and cancer that currently can be pharmaceutically activated either indirectly or by unknown mechanisms. Physiologically, AMPK is activated by AMP or ADP-dependent changes in its accessibility to upstream kinases and phosphatases and by allosteric modulation of its kinase activity by AMP and glycogen. In this project we propose to unravel the structural mechanisms of allosteric regulation of the AMPK kinase activity to provide a structural framework for the rational design of new therapeutic AMPK modulators.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM104212-02
Application #
8703136
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
2013-08-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
2
Fiscal Year
2014
Total Cost
$361,463
Indirect Cost
$146,059
Name
Van Andel Research Institute
Department
Type
DUNS #
129273160
City
Grand Rapids
State
MI
Country
United States
Zip Code
49503
Zhao, Li-Hua; Yin, Yanting; Yang, Dehua et al. (2016) Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors. J Biol Chem 291:15119-30
Rowe, Timothy B; Luo, Zhe-Xi; Ketcham, Richard A et al. (2016) X-ray computed tomography datasets for forensic analysis of vertebrate fossils. Sci Data 3:160040
Golani, Lalit K; Wallace-Povirk, Adrianne; Deis, Siobhan M et al. (2016) Tumor Targeting with Novel 6-Substituted Pyrrolo [2,3-d] Pyrimidine Antifolates with Heteroatom Bridge Substitutions via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthes J Med Chem 59:7856-76
Xu, Ting-Hai; Yan, Yan; Kang, Yanyong et al. (2016) Alzheimer's disease-associated mutations increase amyloid precursor protein resistance to γ-secretase cleavage and the Aβ42/Aβ40 ratio. Cell Discov 2:16026
Zhou, X Edward; Gao, Xiang; Barty, Anton et al. (2016) X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex. Sci Data 3:160021
Tan, M H Eileen; Li, Jun; Xu, H Eric et al. (2015) Androgen receptor: structure, role in prostate cancer and drug discovery. Acta Pharmacol Sin 36:3-23
Zhao, Li-Hua; Zhou, X Edward; Yi, Wei et al. (2015) Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3. Cell Res 25:1219-36
Zhi, Xiaoyong; Zhou, X Edward; He, Yuanzheng et al. (2015) Structural basis for corepressor assembly by the orphan nuclear receptor TLX. Genes Dev 29:440-50
Li, Xiaodan; Wang, Lili; Zhou, X Edward et al. (2015) Structural basis of AMPK regulation by adenine nucleotides and glycogen. Cell Res 25:50-66
Kang, Yanyong; Zhou, X Edward; Gao, Xiang et al. (2015) Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523:561-7

Showing the most recent 10 out of 12 publications