The heterotrimeric AMP-activated protein kinase (AMPK) is a key regulator of cellular energy metabolism. It activates energy-producing pathways and inhibits energy-consuming processes in response to low intracellular ATP levels. AMPK has been implicated in various diseases related to energy metabolism, including type 2 diabetes, obesity, and cancer. AMPK is activated by phosphorylation of the activation loop within its kinase domain, and AMP binding to the γ regulatory domain promotes this phosphorylation and protects the enzyme against dephosphorylation. This study shows that ADP binding to one of the two exchangeable AXP binding sites on the regulatory domain also protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. The crystal structure of active AMPK reveals how the activation loop is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide binding site that mediates protection against dephosphorylation. The study also shows that active AMPK has a significantly tighter binding to ADP than to Mg.ATP, explaining how the enzyme is regulated under physiological conditions where Mg.ATP is more abundant than ADP and AMP. AMPK is regulated by a variety of hormones and plays a role in appetite, glucose, lipid and protein metabolism, cell growth and cell polarity. AMPK is a heterotrimeric complex comprising an α catalytic subunit and two regulatory subunits (β and γ). Activation of AMPK requires phosphorylation of Thr-172, which lies in the activation segment of the N-terminal kinase domain of the α subunit. Phosphorylation of Thr-172 leads to a several hundred fold increase in activity. The study also shows that AMP and ADP provide protection of AMPK from dephosphorylation across a similar range of concentrations. The protective effect of ADP is lost in a Wolff-Parkinson-White syndrome mutation. The study also shows that AMP/ADP binding at the weaker of the two exchangeable sites is responsible for protection against dephosphorylation. The crystal structure of active AMPK reveals that the α-hook structure interacts with the γ subunit at the exchangeable binding site-3, which is responsible for mediating AMP/ADP protection against dephosphorylation. The study also shows that the α-hook region binds into this site in the presence of AMP and predicts that the same situation would occur with ADP. The study proposes a model for how AMP/ADP, but not Mg.ATP, protects AMPK against dephosphorylation. The model suggests that binding of the α-hook acts to restrict the flexibility of the preceding α linker region and promotes the interaction of the kinase domain with the regulatory fragment seen in the crystal structure. This interaction protects Thr-172 against dephosphorylation. The study also shows that the α-hook region binds into this site in the presenceThe heterotrimeric AMP-activated protein kinase (AMPK) is a key regulator of cellular energy metabolism. It activates energy-producing pathways and inhibits energy-consuming processes in response to low intracellular ATP levels. AMPK has been implicated in various diseases related to energy metabolism, including type 2 diabetes, obesity, and cancer. AMPK is activated by phosphorylation of the activation loop within its kinase domain, and AMP binding to the γ regulatory domain promotes this phosphorylation and protects the enzyme against dephosphorylation. This study shows that ADP binding to one of the two exchangeable AXP binding sites on the regulatory domain also protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. The crystal structure of active AMPK reveals how the activation loop is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide binding site that mediates protection against dephosphorylation. The study also shows that active AMPK has a significantly tighter binding to ADP than to Mg.ATP, explaining how the enzyme is regulated under physiological conditions where Mg.ATP is more abundant than ADP and AMP. AMPK is regulated by a variety of hormones and plays a role in appetite, glucose, lipid and protein metabolism, cell growth and cell polarity. AMPK is a heterotrimeric complex comprising an α catalytic subunit and two regulatory subunits (β and γ). Activation of AMPK requires phosphorylation of Thr-172, which lies in the activation segment of the N-terminal kinase domain of the α subunit. Phosphorylation of Thr-172 leads to a several hundred fold increase in activity. The study also shows that AMP and ADP provide protection of AMPK from dephosphorylation across a similar range of concentrations. The protective effect of ADP is lost in a Wolff-Parkinson-White syndrome mutation. The study also shows that AMP/ADP binding at the weaker of the two exchangeable sites is responsible for protection against dephosphorylation. The crystal structure of active AMPK reveals that the α-hook structure interacts with the γ subunit at the exchangeable binding site-3, which is responsible for mediating AMP/ADP protection against dephosphorylation. The study also shows that the α-hook region binds into this site in the presence of AMP and predicts that the same situation would occur with ADP. The study proposes a model for how AMP/ADP, but not Mg.ATP, protects AMPK against dephosphorylation. The model suggests that binding of the α-hook acts to restrict the flexibility of the preceding α linker region and promotes the interaction of the kinase domain with the regulatory fragment seen in the crystal structure. This interaction protects Thr-172 against dephosphorylation. The study also shows that the α-hook region binds into this site in the presence