Abstract
AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes. Many pharmacological AMPK activators, and possibly unidentified physiological metabolites, bind to the allosteric drug and metabolite (ADaM) site at the interface between the kinase domain (KD) in the α-subunit and the carbohydrate-binding module (CBM) in the β-subunit. Here, using double electron-electron resonance (DEER) spectroscopy, we demonstrate that the CBM-KD interaction is partially dissociated and the interface highly disordered in the absence of pharmacological ADaM site activators as inferred from a low depth of modulation and broad DEER distance distributions. ADaM site ligands such as 991, and to a lesser degree phosphorylation, stabilize the KD-CBM association and strikingly reduce conformational heterogeneity in the ADaM site. Our findings that the ADaM site, formed by the KD-CBM interaction, can be modulated by diverse ligands and by phosphorylation suggest that it may function as a hub for integrating regulatory signals.
Highlights
AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes
Using double electron– electron resonance (DEER) spectroscopy, we demonstrate that the carbohydrate-binding module (CBM)–kinase domain (KD) interaction is partially dissociated and the interface highly disordered in the absence of pharmacological allosteric drug and metabolite (ADaM) site activators as inferred from a low depth of modulation and broad DEER distance distributions
It has been reported that AMPK with ␣2 C130A and C174A mutations cannot be activated by energy stress in cells or in vitro [34]
Summary
A cysteine-free version of AMPK is catalytically active and regulated by adenine nucleotides and 991. Cys-free MBP-␣12␥1 AMPK, in which the corresponding ␣1 residues Cys-132 and Cys-176 are mutated to Ala and Ser, respectively, is clearly activated by AMP ( it has reduced catalytic activity) This protein is activated by the ADaM site ligand 991, and not by. For the distal 2K88C–␣1C211 and 2K88C–␣1F225C interactions (Fig. 3, middle and right columns, respectively), 991 and derivatives largely depopulated the dominant DEER distance peak at ϳ41 Å and in the case of 2K88C–␣1C211, 991, and R734 shifted the ϳ57 Å peak by 3–5 Å to a shorter distance These ligands strongly reduce ADaM site heterogeneity, presumably by stabilizing the CBM–KD interaction through their direct interaction with both CBM and KD [15, 16]. ADaM site ligand binding induces a small expansion of the site
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