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 #
5R01GM104212-03
Application #
8831699
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
2015-04-01
Budget End
2016-03-31
Support Year
3
Fiscal Year
2015
Total Cost
$362,224
Indirect Cost
$146,059
Name
Van Andel Research Institute
Department
Type
DUNS #
129273160
City
Grand Rapids
State
MI
Country
United States
Zip Code
49503
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Yin, Wanchao; Zhou, X Edward; Yang, Dehua et al. (2018) Crystal structure of the human 5-HT1B serotonin receptor bound to an inverse agonist. Cell Discov 4:12
Harikumar, Kaleeckal G; Yan, Yan; Xu, Ting-Hai et al. (2017) Bioluminescence Resonance Energy Transfer (BRET) Assay for Determination of Molecular Interactions in Living Cells. Bio Protoc 7:
Pal, Kuntal; Bandyopadhyay, Abhishek; Zhou, X Edward et al. (2017) Structural Basis of TPR-Mediated Oligomerization and Activation of Oncogenic Fusion Kinases. Structure 25:867-877.e3
Yan, Yan; Xu, Ting-Hai; Harikumar, Kaleeckal G et al. (2017) Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the ?-secretase cleavage sites. J Biol Chem 292:15826-15837
Xu, Ting-Hai; Yan, Yan; Harikumar, Kaleeckal G et al. (2017) Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization. Bio Protoc 7:
Gu, Xin; Yan, Yan; Novick, Scott J et al. (2017) Deconvoluting AMP-activated protein kinase (AMPK) adenine nucleotide binding and sensing. J Biol Chem 292:12653-12666
Zhang, Feng; Ke, Jiyuan; Zhang, Li et al. (2017) Structural insights into alternative splicing-mediated desensitization of jasmonate signaling. Proc Natl Acad Sci U S A 114:1720-1725
Yan, Yan; Xu, Ting-Hai; Melcher, Karsten et al. (2017) Defining the minimum substrate and charge recognition model of gamma-secretase. Acta Pharmacol Sin 38:1412-1424
Yin, Yanting; de Waal, Parker W; He, Yuanzheng et al. (2017) Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors. J Biol Chem 292:9865-9881

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