The article investigates the structure and regulation of mammalian AMP-activated protein kinase (AMPK) by adenosine diphosphate (ADP). AMPK plays a crucial role in cellular energy metabolism, activating energy-producing pathways and inhibiting energy-consuming processes in response to low ATP levels. The study reveals that ADP binding to one of the two ATP binding sites on the regulatory domain protects AMPK from dephosphorylation, though it does not lead to allosteric activation. Active AMPK binds ADP more tightly than magnesium adenosine triphosphate (Mg.ATP), which is consistent with physiological conditions where Mg.ATP concentrations are much higher than ADP and AMP. The crystal structure of an active AMPK complex 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. The authors propose a model where AMP/ADP binding at the weaker site shifts the equilibrium between dephosphorylation-sensitive and insensitive states, thus regulating AMPK activity. The study also highlights the importance of the α-hook region in mediating protection against dephosphorylation.The article investigates the structure and regulation of mammalian AMP-activated protein kinase (AMPK) by adenosine diphosphate (ADP). AMPK plays a crucial role in cellular energy metabolism, activating energy-producing pathways and inhibiting energy-consuming processes in response to low ATP levels. The study reveals that ADP binding to one of the two ATP binding sites on the regulatory domain protects AMPK from dephosphorylation, though it does not lead to allosteric activation. Active AMPK binds ADP more tightly than magnesium adenosine triphosphate (Mg.ATP), which is consistent with physiological conditions where Mg.ATP concentrations are much higher than ADP and AMP. The crystal structure of an active AMPK complex 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. The authors propose a model where AMP/ADP binding at the weaker site shifts the equilibrium between dephosphorylation-sensitive and insensitive states, thus regulating AMPK activity. The study also highlights the importance of the α-hook region in mediating protection against dephosphorylation.