Structures of dynamic interactors at native proteasomes by PhIX-MS and cryo-electron microscopy

Area of Science:

• Structural biology
• Proteomics
• Molecular cell biology

Background:

• Molecular machines utilize dynamic, low-affinity interactions for function.
• Understanding these transient interactions is crucial for deciphering cellular mechanisms.

Purpose of the Study:

• To develop a novel structural proteomics workflow, PhIX-MS, for capturing transient protein interactions in cells.
• To apply PhIX-MS and cryo-electron microscopy (cryo-EM) to map protein interactions within the proteasome.

Main Methods:

• Photo-induced in situ crosslinking-mass spectrometry (PhIX-MS) workflow.
• UV-activated crosslinking to capture topological information of transient interactions.
• Integration with cryo-electron microscopy (cryo-EM) and AlphaFold for structural analysis.

Main Results:

• Mapped the redox sensor TXNL1 to the proteasome regulatory particle (RP), near RPN2/PSMD1 and RPN13/ADRM1.
• Resolved structures of RPs bound to TXNL1 and/or the chaperone PSMD5/S5b, revealing structural rearrangements.
• Identified the ubiquitin ligase UBE3C/Hul5 at RPN2, RPN3, and RPN10, tethering it above the substrate entry channel.

Conclusions:

• PhIX-MS enables the localization of native, low-affinity protein interactions within cells.
• The approach is broadly applicable to studying dynamic macromolecular assemblies like the proteasome.
• Revealed the structural context of TXNL1 and UBE3C in proteasome function.