Structures of dynamic interactors at native proteasomes by PhIX-MS and cryoelectron microscopy.

Area of Science:

• Structural Biology
• Proteomics
• Molecular Cell Biology

Background:

• Proteasome function relies on transient protein interactions, which are difficult to study structurally.
• Understanding these interactions is crucial for elucidating proteasome assembly and regulation.

Purpose of the Study:

• To develop and apply a novel structural proteomics workflow, PhIX-MS, to map transient protein interactions within the proteasome.
• To resolve the native, low-affinity interactions of key proteasome-associated proteins.

Main Methods:

• Photo-induced In situ Crosslinking-Mass Spectrometry (PhIX-MS) to stabilize transient interactions in cells.
• Cryo-electron microscopy (cryo-EM) for structural determination.
• AlphaFold modeling for computational structure prediction.

Main Results:

• PhIX-MS mapped the redox sensor TXNL1 to the proteasome regulatory particle (RP), detailing its domain placements.
• The chaperone PSMD5 was resolved bound to the RP, blocking proteolytic core particle (CP) binding.
• Ubiquitin ligase UBE3C/Hul5 was localized to the RP, suggesting coupled activity with RPN11.

Conclusions:

• PhIX-MS successfully captures topological information of transient interactions in situ.
• The study provides high-resolution structural insights into proteasome regulatory mechanisms.
• This integrative approach is broadly applicable to dynamic macromolecular assemblies.