Journal Article

PUBDB-2025-03837

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Structural Heterogeneity of Proteoform-Ligand Complexes in Adenosine Monophosphate-Activated Protein Kinase Uncovered by Integrated Top-Down Mass Spectrometry

Chan, H.-J. ; Krichel, B.Extern*CSSB*DESY* ; Bandura, L. J. ; Chapman, E. A. ; Rogers, H. T. ; Fischer, M. S. ; Roberts, D. S. ; Gao, Z. ; Wang, M.-D. ; Wu, J. ; Uetrecht, C.CSSB*XFEL.EU*DESY* ; Jin, S. ; Ge, Y. (Corresponding author)

2025
ACS Publications Washington, DC

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Please use a persistent id in citations: doi:10.1021/jacs.5c06950

Abstract: Adenosine monophosphate-activated protein kinase (AMPK) is a heterotrimeric complex (αβγ) 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, noncovalent interactions, and higher-order structures of the kinase complex remains poorly understood. Herein, we report for the first time 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, while denatured TDMS comprehensively characterizes the proteoforms and localizes the phosphorylation site. Notably, by integrating native TDMS and AlphaFold, we elucidate a flexibly connected regulatory region of AMPK β subunit that was previously unresolvable with traditional structural biology tools. Our findings offer new perspectives on protein kinase regulation and establish a versatile framework for comprehensive characterization of proteoform-ligand complexes.

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Contributing Institute(s):

1. CSSB - Leibniz-Institut für Experimentelle Virologie (LIV) / DESY - Charlotte Uetrecht (CSSB-LIV/DESY-CU)

Research Program(s):

1. 633 - Life Sciences – Building Blocks of Life: Structure and Function (POF4-633) (POF4-633)
2. Top-AMPK - Putting the top down on AMPK protein complex (101068151) (101068151)
3. SPOCkS MS - Sampling Protein cOmplex Conformational Space with native top down Mass Spectrometry (759661) (759661)

Experiment(s):

1. No specific instrument

Appears in the scientific report 2025

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Database coverage:
; BIOSIS Previews ; Biological Abstracts ; Chemical Reactions ; Clarivate Analytics Master Journal List ; Current Contents - Life Sciences ; Current Contents - Physical, Chemical and Earth Sciences ; Ebsco Academic Search ; Essential Science Indicators ; IF >= 15 ; Index Chemicus ; JCR ; Nationallizenz ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection

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