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Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength)	Production and Crystallization of Full-Length Human AMP-Activated Protein Kinase (α1β1γ1) Julia A. Hubbard , Bing Xiao , Jon R. Wilson DOI: 10.1007/978-1-4939-7598-3_1 Diamond Proposal Number(s): [9826] Show Abstract ▼ Abstract: Determination of the crystal structure of AMP-activated protein kinase (AMPK) is fundamental to understanding its biological function and role in a number of diseases related to energy metabolism including type 2 diabetes, obesity, and cancer. We describe methods for the expression and purification of a human full-length active AMPK complex that is suitable for biochemical and structural analyses, followed by methods for its crystallization in complex with small molecule activators. Quality control of the purified protein by functional and biophysical analysis was an essential part of the process enabling the achievement of crystals of the full-length protein capable of being used for high-resolution structure determination by X-ray diffraction. X-ray structures have been determined of both phosphorylated and non-phosphorylated forms of full-length human AMPK α1β1γ1.	Feb 2018
I02-Macromolecular Crystallography	ADP regulates SNF1, the Saccharomyces cerevisiae homolog of AMP-activated protein kinase Faith V. Mayer , Richard Heath , Elizabeth Underwood , Matthew J. Sanders , David Carmena , Rhonda R. Mccartney , Fiona C. Leiper , Bing Xiao , Chun Jing , Philip A. Walker , Lesley F. Haire , Roksana Ogrodowicz , Stephen R. Martin , Martin C. Schmidt , Steven J. Gamblin , David Carling Cell Metabolism 14 (5), 707 - 714 DOI: 10.1016/j.cmet.2011.09.009 PMID: 22019086 Diamond Proposal Number(s): [7707] Open Access Show Abstract ▼ Abstract: The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.	Nov 2011
I03-Macromolecular Crystallography	Structure of mammalian AMPK and its regulation by ADP Bing Xiao , Matthew J. Sanders , Elizabeth Underwood , Richard Heath , Faith V. Mayer , David Carmena , Chun Jing , Phil Walker , John F. Eccleston , Lesley F. Haire , Peter Saiu , Steven A. Howell , Rein Aasland , Stephen R. Martin , David Carling , Steven Gamblin Nature 472, 230 - 233 DOI: 10.1038/nature09932 PMID: 21399626 Diamond Proposal Number(s): [1228] Show Abstract ▼ Abstract: The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes1. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer2, 3, 4, 5, 6. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain7: AMP binding to the ?-regulatory domain promotes phosphorylation by the upstream kinase8, protects the enzyme against dephosphorylation, as well as causing allosteric activation9. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.	Mar 2011

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