Structures of dynamic interactors at native proteasomes by PhIX-MS and cryo-electron microscopy.
Molecular machines rely on dynamic, low-affinity interactions to perform their functional roles. We developed PhIX-MS (photo-induced in situ crosslinking-mass spectrometry), a structural proteomics workflow to capture topological information for such transient interactions in cells by UV-activated crosslinking. Applying PhIX-MS with cryo-electron microscopy (cryo-EM) to proteasomes, we mapped the redox sensor ... TXNL1 at the proteasome regulatory particle (RP), including its dynamic thioredoxin-like domain near RPN2/PSMD1 and RPN13/ADRM1, where it is ideal for reducing substrates prior to proteolysis. RPs without the proteolytic core particle (CP) were structurally resolved while bound to TXNL1 and/or the chaperone PSMD5/S5b, which inserts its C terminus into the ATPase pore, causing extensive structural rearrangements. Additionally, PhIX-MS and AlphaFold identified the ubiquitin ligase UBE3C/Hul5 at RPN2, RPN3, and a dynamic RPN10 region, tethering UBE3C above the substrate entry channel. Our integrative approach enables the localization of native, low-affinity protein interactions and is broadly applicable to dynamic macromolecular assemblies.
Mesh Terms:
Adenosine Triphosphatases, Biotinylation, Cryoelectron Microscopy, HCT116 Cells, Humans, Mass Spectrometry, Models, Molecular, Models, Structural, Proteasome Endopeptidase Complex, Protein Conformation, Protein Interaction Maps, Thioredoxins, Ubiquitin-Protein Ligases
Adenosine Triphosphatases, Biotinylation, Cryoelectron Microscopy, HCT116 Cells, Humans, Mass Spectrometry, Models, Molecular, Models, Structural, Proteasome Endopeptidase Complex, Protein Conformation, Protein Interaction Maps, Thioredoxins, Ubiquitin-Protein Ligases
Mol Cell
Date: Aug. 06, 2026
PubMed ID: 42476128
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