Deconvoluting AMP-activated protein kinase (AMPK) adenine nucleotide binding and sensing

Abstract

AMP-activated protein kinase (AMPK) is a central cellular energy sensor that adapts metabolism and growth to the energy state of the cell. AMPK senses the ratio of adenine nucleotides (adenylate energy charge) by competitive binding of AMP, ADP, and ATP to three sites (CBS1, CBS3, and CBS4) in its γ-subunit. Because these three binding sites are functionally interconnected, it remains unclear how nucleotides bind to individual sites, which nucleotides occupy each site under physiological conditions, and how binding to one site affects binding to the other sites. Here, we comprehensively analyze nucleotide binding to wild-type and mutant AMPK protein complexes by quantitative competition assays and by hydrogen-deuterium exchange MS. We also demonstrate that NADPH, in addition to the known AMPK ligand NADH, directly and competitively binds AMPK at the AMP-sensing CBS3 site. Our findings reveal how AMP binding to one site affects the conformation and adenine nucleotide binding at the other two sites and establish CBS3, and not CBS1, as the high affinity exchangeable AMP/ADP/ATP-binding site. We further show that AMP binding at CBS4 increases AMP binding at CBS3 by 2 orders of magnitude and reverses the AMP/ATP preference of CBS3. Together, these results illustrate how the three CBS sites collaborate to enable highly sensitive detection of cellular energy states to maintain the tight ATP homeostastis required for cellular metabolism.

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

“…S11). These results confirm the stable binding of AMP at CBS4 as well as the recent reassignment of CBS3 and CBS1 as the exchangeable higher-and lower-affinity nucleotide binding-sites, respectively (35).…”

Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK)

“…First, the ADaM site ligand pocket is adaptable and its shape and size is modulated by ligand binding. For instance, we have calculated a pocket size of 486.5 Å 3 for AMPK ␣ 2 ␤ 1 ␥ 1 bound to 991 (PDB 4CFE); pocket sizes of 374.5 Å 3 and 474.9 Å 3 , respectively, for AMPK ␣ 1 ␤ 1 ␥ 1 bound to the A769662 derivatives Cl-A769662 (PDB 4QFR) and Br 2 -A769662 (PDB 4QFS); pocket sizes of 547.1 Å 3 and 522.7 Å 3 , respectively, for AMPK ␣ 1 ␤ 1 ␥ 1 bound to the 991 derivatives R734 and R739; 4 and pocket sizes of 195.0 and 396.1 Å 3 , respectively, for apo-AMPK ␣ 1 ␤ 1 ␥ 1 (PDB 4QFG) and apo-AMPK ␣ 1 ␤ 2 ␥ 1 (PDB 4RER). Second, the first identified ADaM site agonist, the thienopyridone A769662, only activates ␤ 1 -subunit containing complexes, requires phosphorylation of Ser-108 of the CBM, and can synergize with AMP to fully activate AMPK even in the absence of AMPK activation loop phosphorylation (27,28).…”

“…Third, a 991-derivative AMPK activator, R739, selectively and robustly increases protection of the activation loop of ␤ 2 -containing AMPK against dephosphorylation without increasing direct allosteric AMPK activation. 4 Fourth, the A769662 derivative MT47-100 is an agonist of ␤ 1 -containing complexes, but an inhibitor of ␤ 2 -containing complexes (30). Fifth, the ADaM site-forming KD-CBM interaction is destabilized by glycogen, which binds the CBM distal to the ADaM site, and is stabilized by CBM phosphorylation (13), whereas adenine nucleotides constrain the CBM flexibility (4).…”

“…4 Fourth, the A769662 derivative MT47-100 is an agonist of ␤ 1 -containing complexes, but an inhibitor of ␤ 2 -containing complexes (30). Fifth, the ADaM site-forming KD-CBM interaction is destabilized by glycogen, which binds the CBM distal to the ADaM site, and is stabilized by CBM phosphorylation (13), whereas adenine nucleotides constrain the CBM flexibility (4). Collectively, this indicates that ADaM site modulation through AMPK ligands is highly complex and likely proceeds through adoption of different ensembles of conformations in solution.…”

AMPK hierarchy: a matter of space and time

“…Several lines of evidence suggest that one of these sites binds AMP with high affinity (low μM) and the other with much lower affinity (high μM). 10 Based on current knowledge, the nucleotide binding properties of the two sites do not provide an obvious mechanistic explanation for the axin-dependency of AMPK activation at different AMP levels. Finally, the study makes the important observation that activation of Published online: 25 April 2019 distinct subcellular pools of AMPK can lead to differential phosphorylation of AMPK substrates.…”