Structures of AMP-activated protein kinase bound to novel pharmacological activators in phosphorylated, non-phosphorylated, and nucleotide-free states

Abstract

Edited by Wolfgang Peti 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 ␣-subunit and the carbohydratebinding module (CBM) of the ␤-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 ␤ 2-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant ␣ 1 ␤ 2 ␥ 1 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 ␥-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 ␣ 1 ␤ 1 ␥ 1 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. AMPK 2 is a three-subunit protein kinase that consists of two alternative kinase domain-containing ␣-subunits (␣ 1 and ␣ 2), two alternative carbohydrate-binding module-containing ␤-subunits (␤ 1 and ␤ 2), and three alternative AMP/ADP/ATPbinding ␥-subunits (␥ 1 , ␥ 2 , and ␥ 3) (1-3). In humans, the ␤ 1 isoform is only present in a fraction of AMPK complexes in liver but not immunologically detectable in skeletal muscle, whose predominant isoform is ␣ 2 ␤ 2 ␥ 1 (4-6). AMPK senses the energy state of cells by competitive binding of AMP, ADP, and ATP to three separate sites in its ␥-subunit. Upon energy stress, i.e. increases in the ratio of AMP and ADP to ATP, AMPK is activated ϳ100-fold by activation loop phosphorylation and an additional 2-10-fold by direct allosteric kinase activation (7). The activated enzyme phosphorylates key metabolic and regulatory proteins to stimulate ATP-generating catabolic pathways and inhibit ATP-consuming anabolic pathways and cellular programs (8-10). AMPK is an attractive potential target for the treatment of metabolic diseases, most prominently type 2 diabetes (9, 11, 12). Currently, metformin is the frontline drug for the treatment of type 2 diabetes. Metformin is taken up predominantly by the liver where it generates AMPK-activating energy stress by mildly inhibiting oxidative phosphorylation. In addition, it has been reported that at low pharmacological levels AMPK increases phosphorylation through LKB1 by inducing AMPK heterotrimer formation (13) and formation of an LKB1-This work was suppo...

AMPK and TOR: The Yin and Yang of Cellular Nutrient Sensing and Growth Control

“…Another ligand-binding site lies in a cleft (termed the ADaM site) between the other face of the CBM (i.e., opposite to the glycogen-binding site) and the N-lobe of the kinase domain on the a subunit (Figure 2). Several ligands that bind in this site cause a dramatic allosteric activation of AMPK with, usually, a more modest effect to promote net Thr172 phosphorylation (Gö ransson et al, 2007;Sanders et al, 2007;Scott et al, 2014;Yan et al, 2019). However, a curious feature is that with the exception of salicylate (a natural product of plants, but not animals) (Hawley et al, 2012), all of the compounds currently known to bind there are synthetic molecules that emerged from high-throughput screens searching for allosteric activators of AMPK (e.g., Cokorinos et al, 2017;Cool et al, 2006;Myers et al, 2017).…”

Drug resistance in diploid yeast is acquired through dominant alleles, haploinsufficiency, gene duplication and aneuploidy

“… A . The structure the mammalian AMPK (6c9h.pdb) bound to the activator R734 [ 46 ] is shown as a model for the Snf1 kinase heterotrimer. The alpha subunit is shown in green, the beta subunit in blue and the gamma subunit in cyan.…”

Nutrient Signaling and Lysosome Positioning Crosstalk Through a Multifunctional Protein, Folliculin

“…The α subunit contains a typical serine/threonine protein kinase catalytic domain (Hanks et al, 1988) as well as several phosphorylation sites, with α1/α2-Thr172/174 being essential for AMPK activity (Stein et al, 2000) (Figure 2A). Multiple crystal structures of the complete kinase complex support a functional role for Thr172/174: binding of AMP or other synthetic activators protects this threonine residue from dephosphorylation and, therefore, AMPK inactivation through phosphatase activity (Figure 2B, inset) (Xiao et al, 2011(Xiao et al, , 2013Calabrese et al, 2014;Yan et al, 2019). Two upstream kinases, Liver kinase B1 (LKB1) and calmodulin kinase kinase (CAMKK), are responsible for the activation of AMPK by phosphorylation of Thr172/174, depending on the cellular context (Woods et al, 2003;Shaw et al, 2004).…”