MassIVE MSV000097430

Partial Public PXD062177

Structural Heterogeneity of Proteoform-Ligand Complexes in AMP-Activated Protein Kinase Uncovered by Integrated Top-Down Mass Spectrometry

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Description

AMP-activated protein kinase (AMPK) is a heterotrimeric complex 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 remain 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 provides valuable insights into the higher-order structure of AMPK, subunit composition, and AMP binding stoichiometry, whilst denatured TDMS characterizes the proteoforms and localizes the phosphorylation site. This is the first study to structurally characterize AMPK proteoform-ligand complexes. Notably, using native TDMS and AlphaFold, we resolve a flexible regulatory region that has been difficult to visualize with traditional structural biology tools. These findings offer new perspectives on protein kinase regulation and establish a powerful framework for elucidating other kinase complexes. [doi:10.25345/C5VX06G0B] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: top-down mass spectrometry ; native top-down mass spectrometry ; AMP-activated protein kinase ; post-translational modifications ; protein-ligand binding ; DatasetType:Proteomics

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Principal Investigators:
(in alphabetical order) 		Ying Ge, University of Wisconsin-Madison, United States of America
Submitting User: rubyhjchan
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