Chan, H.-J.; Krichel, B.; Bandura, L. J.; Chapman, E. A.; Rogers, H. T.; Fischer, M. S.; Roberts, D. S.; Gao, Z.; Wang, M.-D.; Wu, J.; Uetrecht, C.; Jin, S.; Ge, Y.

AMP-activated protein kinase (AMPK) is a heterotrimeric complex ({beta}{gamma}) that serves as a master regulator of cellular metabolism, making it a prominent drug target for various diseases. Post-translational modifications (PTMs) and ligand binding significantly affect the activity and function of AMPK. However, the dynamic interplay of PTMs, non-covalent interactions, and higher-order structures of the kinase complex remains poorly understood. Herein, we report the structural heterogeneity of the AMPK complex arising from ligand binding and proteoforms--protein products derived from PTMs, alternative splicing, and genetic variants--using integrated native and denatured top-down mass spectrometry (TDMS). The fully intact AMPK heterotrimeric complex exhibits heterogeneity due to phosphorylation and multiple adenosine monophosphate (AMP) binding states. Native TDMS delineates the subunit composition, AMP binding stoichiometry, and higher-order structure of AMPK complex, whilst denatured TDMS comprehensively characterizes the proteoforms and localizes the phosphorylation site. This is the first study to structurally characterize AMPK proteoform-ligand complexes. Notably, by integrating native TDMS and AlphaFold, we elucidate a flexibly connected regulatory region of AMPK {beta} subunit that has been difficult to visualize with traditional structural biology tools. Our findings uncover previously unresolvable structural features of AMPK, offer new perspectives on protein kinase regulation, and establish a versatile framework for comprehensive characterization of proteoform-ligand complexes.