Elucidating the Activation Mechanism of AMPK by Direct Pan-Activator PF-739.

Adenosine monophosphate-activated protein kinase (AMPK) is a key energy sensor regulating the cell metabolism in response to energy supply and demand. The evolutionary adaptation of AMPK to different tissues is accomplished through the expression of distinct isoforms that can form up to 12 heterotrimeric complexes, which exhibit notable differences in the sensitivity to direct activators. To comprehend the molecular factors of the activation mechanism of AMPK, we have assessed the changes in the structural and dynamical properties of ß1- and ß2-containing AMPK complexes formed upon binding to the pan-activator PF-739. The analysis revealed the molecular basis of the PF-739-mediated activation of AMPK and enabled us to identify distinctive features that may justify the slightly higher affinity towards the ß1-isoform, such as the ß1-Asn111 to ß2-Asp111 substitution, which seems to be critical for modulating the dynamical sensitivity of ß1- and ß2 isoforms. The results are valuable in the design of selective activators to improve the tissue specificity of therapeutic treatment.

Structural basis of the selective activation of enzyme isoforms: Allosteric response to activators of ß1- and ß2-containing AMPK complexes.

AMP-activated protein kinase (AMPK) is a key energy sensor regulating the cell metabolism in response to energy supply and demand. The evolutionary adaptation of AMPK to different tissues is accomplished through the expression of distinct isoforms that can form up to 12 complexes, which exhibit notable differences in the sensitivity to allosteric activators. To shed light into the molecular determinants of the allosteric regulation of this energy sensor, we have examined the structural and dynamical properties of ß1- and ß2-containing AMPK complexes formed with small molecule activators A-769662 and SC4, and dissected the mechanical response leading to active-like enzyme conformations through the analysis of interaction networks between structural domains. The results reveal the mechanical sensitivity of the α2ß1 complex, in contrast with a larger resilience of the α2ß2 species, especially regarding modulation by A-769662. Furthermore, binding of activators to α2ß1 consistently promotes the pre-organization of the ATP-binding site, favoring the adoption of activated states of the enzyme. These findings are discussed in light of the changes in the residue content of ß-subunit isoforms, particularly regarding the ß1Asn111 â†’ ß2Asp111 substitution as a key factor in modulating the mechanical sensitivity of ß1- and ß2-containing AMPK complexes. Our studies pave the way for the design of activators tailored for improving the therapeutic treatment of tissue-specific metabolic disorders.