Figure 7.

Changes in HDX-MS protection in the presence of AXP mixtures mimicking non-stress and energy stress conditions. A, AMPK kinase assay in the presence of AXP mixtures mimicking nonstress (4.5 mm ATP, 0.4 mm ADP, 0.04 mm AMP) and energy stress (3.8 mm ATP, 1.0 mm ADP, 0.3 mm AMP) conditions. Left panel, AlphaScreen-based kinase assay; right panel, radioactive kinase assay. B, HDX-MS perturbation map of AMPK in the presence of the two different AXP mixtures. HDX-MS changes were overlaid as heat map onto the structure of α1β2γ1 AMPK (PDB code 4RER). Main areas of differential protection are highlighted by dashed outlines. Note that there is no change at the catalytic site, indicating that the catalytic site is constitutively bound by ATP as expected. The color code (% change) is shown below the structures. The relatively mild changes are consistent with noticeable ATP exchange only at CBS3, whereas the non-physiological shift from high concentration of pure AMP to high concentration of pure ATP resulted in strong HDX changes (Fig. 6A), consistent with near full nucleotide exchange at both CBS3, CBS1, and ATP binding/release at the catalytic site.