Purification and Phosphorylation of AMPK Complexes

AMPK complexes were expressed in E. coli BL21 (DE3) cells, purified by affinity chromatography using nickel-Sepharose and phosphorylated by incubation with CaMKKβ as described previously9 (link). AMPK activity was determined using 0.2 mM SAMS peptide9 (link), 0.2 mM ATP and 5 mM MgCl₂. Dephosphorylation of AMPK by recombinant PP2Cα was monitored either by measuring AMPK activity using the SAMS peptide assay or by Western blotting of phospho-T172. Western blot signals for phospho-T172 and total AMPK α subunit (determined using sheep anti-α1 or anti-α2 antibodies) were quantified using the Li-Cor Odyssey infrared imaging system. Uncorrected fluorescence spectra of the nucleotides (3′-(7-diethylaminocoumarin-3-carbonylamino)-3′-deoxy-ADP (C-ADP), and 3′-(7-diethylaminocoumarin-3 carbonylamino)-3′-deoxy-ATP (C-ATP) (both generous gifts from Dr. Martin Webb, MRC NIMR, London)) and NADH and their complexes were recorded at 20°C using a Jasco FP-6300 fluorimeter. Binding of nucleotides was monitored by titrating nucleotide (4-10 μM) with AMPK. Dissociation constants for AMP, ADP, and ATP were determined using competition assays. The engineered crystallization construct was expressed as a His-tag fusion protein in E. coli. Purified protein was phosphorylated using CAMKKβ before mixing with AMP and staurosporine. Crystals were grown by the hanging drop method using isopropanol and MPD as precipitant. Diffraction data were collected on the Diamond Light Source, Oxford and processed using Denzo and Scalepack28 . The structure was solved by molecular replacement using Amore29 and standard refinement was carried out with Refmac530 (link) with manual model building with COOT. Figures were created with Pymol (http://pymol.sourceforge.net/).