Fine tuning energy metabolism in skeletal muscle: Discovery of a novel autoinhibitory mechanism in the N-terminal extension of AMPKγ3

<jats:p>AMP-activated protein kinase (AMPK) regulates metabolism in response to metabolic stress that includes stimulating glucose uptake in skeletal muscle independently of the canonical insulin signalling pathway, positioning it as an attractive therapeutic target for insulin resistance and type 2 diabetes mellitus (T2DM). AMPK is an αβγ heterotrimer, with multiple isoforms for each subunit enabling the formation of 12 different complexes with distinct tissue expression profiles. Among these, the α2β2γ3 complex is predominantly expressed in skeletal muscle, the major site of glucose disposal and a highly desirable therapeutic target for T2DM. Here, we characterise the functional role of a unique, 182 residue N-terminal extension (NTE) within γ3 subunit. Deletion of the γ3-NTE from α2β2γ3 complex increases basal AMPK activity without affecting activation by AMP or pharmacological AMPK activators, demonstrating the γ3-NTE performs an autoinhibitory function. Using complementary biophysical techniques, including hydrogen-deuterium exchange-mass spectrometry, surface plasmon resonance, chemical crosslinking and co-pulldowns, we identified a 39-residue sequence in the γ3-NTE (residues 129-168), that directly interacts with the αC-helix of the AMPK kinase domain small lobe, a key regulatory element in many protein kinases. Using AlphaFold3, we probe the interaction predicted to take place between a γ3-NTE α-helix (γ3-iHelix; ~T142-E154) and the αC-helix in the α2β2γ3 complex. These findings provide the groundwork for developing novel T2DM therapies that target AMPK activation selectively in skeletal muscle involving reversal of the γ3 autoinhibition.</jats:p>