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.
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References
Xiao B, Heath R, Saiu P, Leiper FC, Leone P, Jing C, Walker PA, Haire L, Eccleston JF, Davis CT, Martin SR, Carling D, Gamblin SJ (2007) Structural basis for AMP binding to mammalian AMP-activated protein kinase. Nature 449:496–500
Xiao B, Sanders MJ, Underwood E, Heath R, Mayer FV, Carmena D, Jing C, Walker PA, Eccleston JF, Haire LF, Saiu P, Howell SA, Aasland R, Martin SR, Carling D, Gamblin SJ (2011) Structure of mammalian AMPK and its regulation by ADP. Nature 472:230–233
Underwood EA (2013) Nucleotide regulation of AMP-activated protein kinase. Doctoral thesis, University College London
Xiao B, Sanders MJ, Carmena D, Bright NJ, Haire LF, Underwood E, Patel BR, Heath RB, Walker PA, Hallen S, Giordanetto F, Martin SR, Carling D, Gamblin SJ (2013) Structural basis of AMPK regulation by small molecule activators. Nat Commun 4:3017
Calabrese MF, Rajamohan F, Harris MS, Caspers NL, Magyar R, Withka JM, Wang H, Borzilleri KA, Sahasrabudhe PV, Hoth LR, Geoghegan KF, Han S, Brown J, Subashi TA, Reyes AR, Frisbie RK, Ward J, Miller RA, Landro JA, Londregan AT, Carpino PA, Cabral S, Smith AC, Conn EL, Cameron KO, Qiu X, Kurumbail RG (2014) Structural basis for AMPK activation: natural and synthetic ligands regulate kinase activity from opposite poles by different molecular mechanisms. Structure 22:1161–1172
Langendorf CG, Ngoei KR, Scott JW, Ling NX, Issa SM, Gorman MA, Parker MW, Sakamoto K, Oakhill JS, Kemp BE (2016) Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding. Nat Commun 7:10912
Salatto CT, Miller RA, Cameron KO, Cokorinos E, Reyes A, Ward J, Calabrese MF, Kurumbail RG, Rajamohan F, Kalgutkar AS, Tess DA, Shavnya A, Genung NE, Edmonds DJ, Jatkar A, Maciejewski BS, Amaro M, Gandhok H, Monetti M, Cialdea K, Bollinger E, Kreeger JM, Coskran TM, Opsahl AC, Boucher GG, Birnbaum MJ, DaSilva-Jardine P, Rolph T (2017) Selective activation of AMPK beta1-containing isoforms improves kidney function in a rat model of diabetic nephropathy. J Pharmacol Exp Ther 361:303–311
Li X, Wang L, Zhou XE, Ke J, de Waal PW, Gu X, Tan MH, Wang D, Wu D, Xu HE, Melcher K (2015) Structural basis of AMPK regulation by adenine nucleotides and glycogen. Cell Res 25:50–66
Leung L-H, Yao X-J, Wong KW, Liu L, Chen X (2016) Identification of a new AMPK activator for treatment of lung cancer. US Patent US9364469(B1)
Woods A, Dickerson K, Heath R, Hong SP, Momcilovic M, Johnstone SR, Carlson M, Carling D (2005) Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab 2:21–33
Xiao B, Hubbard JA, Gamblin SJ (2017) Structure of full length human AMPK (non-phosphorylated at t-loop) in complex with a small molecule activator, a benzimidazole derivative (991). Protein Database: 5ISO
Acknowledgments
This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council and the Wellcome Trust. Julia Hubbard is a recipient of a Daphne Jackson Research Fellowship, which is funded by the Royal Society of Chemistry and the UK Medical Research Council. We greatly acknowledge Diamond Light Source for access to synchrotron time under proposal MX9826.
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Hubbard, J.A., Xiao, B., Wilson, J.R. (2018). Production and Crystallization of Full-Length Human AMP-Activated Protein Kinase (α1β1γ1). In: Neumann, D., Viollet, B. (eds) AMPK. Methods in Molecular Biology, vol 1732. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7598-3_1
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DOI: https://doi.org/10.1007/978-1-4939-7598-3_1
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