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 (photo-induced in situ crosslinking-mass spectrometry), for capturing transient protein interactions within cells.
• To apply PhIX-MS in conjunction with cryo-electron microscopy (cryo-EM) to investigate the structural organization of proteasome complexes.

Main Methods:

• Photo-induced in situ crosslinking-mass spectrometry (PhIX-MS) using UV activation.
• Cryo-electron microscopy (cryo-EM) for high-resolution structural determination.
• AlphaFold for protein structure prediction and analysis.

Main Results:

• PhIX-MS mapped the redox sensor TXNL1 to the proteasome regulatory particle (RP), near RPN2/PSMD1 and RPN13/ADRM1, highlighting its role in substrate reduction.
• Structural resolution of RPs bound to TXNL1 and the chaperone PSMD5/S5b revealed significant structural rearrangements induced by PSMD5.
• Identification of the ubiquitin ligase UBE3C/Hul5 at specific RP subunits (RPN2, RPN3, RPN10), positioning it above the substrate entry channel.

Conclusions:

• The PhIX-MS workflow effectively localizes native, low-affinity protein interactions in cellular contexts.
• The study provides novel structural insights into the dynamic interactions within proteasome complexes, including the roles of TXNL1 and UBE3C.
• This integrative approach is broadly applicable to studying dynamic macromolecular assemblies and their functions.