AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and has attracted significant attention as a therapeutic target for treating metabolic disorders. AMPK activity is stimulated more than 100-fold by phosphorylation of threonine 172 (Thr¹⁷²). Binding of AMP to the γ subunit allosterically activates the kinase. Additionally, many small molecules, e.g. 991, have been identified that bind between the kinase domain and the carbohydrate-binding module of the β subunit, stabilising their interaction and leading to activation. It was reported recently that non-phosphorylated Thr¹⁷² AMPK is activated by AMP and A769662. We present here the crystal structure of non-phosphorylated Thr¹⁷² AMPK in complex with AMP and 991. This structure reveals that the activation loop, as well as the complex overall, is similar to the Thr¹⁷² phosphorylated complex. We find that in the presence of AMP and 991 non-phosphorylated Thr¹⁷², AMPK is much less active than the Thr¹⁷² phosphorylated enzyme. In human cells, the basal level of Thr¹⁷² phosphorylation is very low (∼1%), but is increased 10-fold by treatment with 2-deoxyglucose. In cells lacking the major Thr¹⁷² kinases, LKB1 and CaMKKβ, Thr¹⁷² phosphorylation is almost completely abolished, and AMPK activity is virtually undetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr¹⁷² AMPK can induce an ordered, active-like, conformation of the activation loop explaining how AMPK activity can be measured in vitro without Thr¹⁷² phosphorylation. However, in a cellular context, phosphorylation of Thr¹⁷² is critical for significant activation of AMPK.

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