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

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.

Figure 1.. Characterization of the AMPK heterotrimeric complex by integrated top-down mass spectrometry (TDMS).

(A) AMPK α1β2γ1 heterotrimer was characterized by integrated native and denatured TDMS. Native AMPK complex or denatured subunits were electrosprayed into mass spectrometers. An ultrahigh-resolution FTICR-MS was used for both native and denatured analysis, while a Q-TOF coupled with online RPLC was used for denatured subunit proteoform profiling. (B) The heterogeneous AMPK proteoform-ligand complexes were resolved by native TDMS. (C) Structural characterization of the AMPK complex using native top-down, complex-up, and complex-down analyses. (D) Proteoform characterization was enabled by denatured TDMS, providing sequence and PTM site localization information. PDB: 7M74, AlphaFold: O43741-F1-v4. P: phosphorylation, AMP: adenosine monophosphate.

Figure 2.. Native MS analysis using an FTICR-MS demonstrates the heterogeneity of AMPK heterotrimeric complex.

(A) Native mass spectrum of AMPK αβγ complex. The inset shows a zoomed-in view of the three most abundant charge states z = 25–27+. (B) Deconvoluted native mass spectrum. Six peaks between 153 kDa and 155 kDa attributed to the AMPK complex are labeled with their corresponding proteoforms and bound ligands (P: phosphorylation, AMP: adenosine monophosphate). (C) Relative abundance of the six AMPK proteoform-ligand complexes. Data are presented as mean ± standard deviation (n = 3).

Figure 3.. Complex-up analysis maps the PTMs and ligand binding to specific subunits.

(A) Representative mass spectrum from complex-up analysis shows AMPK heterotrimer dissociated into monomers and heterodimers. Zoomed-in spectrum shows the charge state distribution of ejected (B) β and γ monomers as well as (C) αγ and αβ dimers. Deconvoluted mass spectrum of dissociated (D) β, (E) γ, (F) αγ, and (G) αβ subunits. P: phosphorylation, AMP: adenosine monophosphate.

Figure 4.. Complex-down analysis of ejected β and γ subunits provides sequence information.

Complex-down mass spectra of the ejected (A) β (z = 12+) and (B) γ (z = 16+) subunit precursors. Representative fragment spectra of (C) β and (D) γ subunits. The isotopic fitting is shown in red circles and mass errors are reported. Structural representation of (E) β (AlphaFold: AF-O43741-F1-v4) and (F) γ (PDB: 7M74) labeled with fragments in complex-down analysis. The regions covered by identified fragments are labeled red. NTD: N-terminal domain; CTD: C-terminal domain; CBS: cystathionine beta-synthase motifs.

Figure 5.. Native top-down ECD analysis uncovers a previously unresolved AMPK flexible region.

(A) Representative mass spectrum of native top-down analysis using ECD shows AMPK heterotrimer charge-reduced species and ECD fragments. (B) Selected fragment spectra of three AMPK subunits. The isotopic fitting is shown in red circles and mass errors are reported. (C) AMPK structures annotated with ECD fragmentation sites. A chimeric model was constructed by aligning a cryo-EM structure of AMPK heterotrimeric complex (PDB: 7M74) and a predicted structure of full-length β subunit (AlphaFold: AF-O43741-F1-v4). Experimental and predicted structure of the β subunit is labeled in dark blue and cyan, respectively. Bond cleavage sites are labeled in red. CBM: carbohydrate-binding module, αγ-SBS: αγ subunit-binding sequence. Different domains are labeled with their corresponding amino acid numbers (CBM:1–83, β-linker: 84–112, αγ-SBS: 113–197).

Figure 6.. Denatured TDMS analysis localizes the phosphorylation site in AMPK β subunit.

(A) Deconvoluted mass spectra of the AMPK β proteoforms acquired using ultrahigh-resolution FTICR-MS to achieve isotopic resolution. AMPKβ: unphosphorylated, AMPKβp: monophosphorylated. Structural representation of the β subunit (AlphaFold: AF-O43741-F1-v4) with the experimentally defined phosphorylation site highlighted as a red sphere. The isotopic fitting is shown in red circles and mass errors are reported. (B) Representative CAD and ECD fragment spectra. (C) Sequence maps of the β subunit annotated with identified CAD and ECD fragments.