5'-AMP-activated protein kinase subunit beta-1
cellular response to nutrient levels
Structures of AMP-activated protein kinase bound to novel pharmacological activators in phosphorylated, non-phosphorylated, and nucleotide-free states.
AMP-activated protein kinase (AMPK) is an attractive therapeutic target for managing metabolic diseases. A class of pharmacological activators, including Merck 991, binds the AMPK ADaM site, which forms the interaction surface between the kinase domain (KD) of the α-subunit and the carbohydrate-binding module (CBM) of the β-subunit. Here, we report the development of two new 991-derivative compounds, R734 and R739, which potently activate AMPK in a variety of cell types, including β<sub>2</sub>-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant α<sub>1</sub>β<sub>2</sub>γ<sub>1</sub> isoform yet robustly enhance protection against activation loop dephosphorylation. This mode of activation is reminiscent of that of ADP, which activates AMPK by binding to the nucleotide-binding sites in the γ-subunit, more than 60 Å away from the ADaM site. To understand the mechanisms of full and partial AMPK activation, we determined the crystal structures of fully active phosphorylated AMPK α<sub>1</sub>β<sub>1</sub>γ<sub>1</sub> bound to AMP and R734/R739 as well as partially active nonphosphorylated AMPK bound to R734 and AMP and phosphorylated AMPK bound to R734 in the absence of added nucleotides at <3-Å resolution. These structures and associated analyses identified a novel conformational state of the AMPK autoinhibitory domain associated with partial kinase activity and provide new insights into phosphorylation-dependent activation loop stabilization in AMPK.