Comparative Structural Analysis of 20S Proteasome Ortholog Protein Complexes by Native Mass Spectrometry

Area of Science:

• Biochemistry
• Structural Biology
• Evolutionary Biology

Background:

• Ortholog protein complexes perform similar functions across species but evolve structural variations due to adaptation.
• High-resolution structures are often unavailable, hindering the study of these variations.
• The 20S proteasome, a key protein degradation machine, serves as an excellent model for studying evolutionary changes in protein complexes.

Purpose of the Study:

• To develop and apply a multilevel experimental approach using native mass spectrometry (MS) to investigate structural preservation and variations in highly related protein complexes.
• To elucidate the evolutionary trajectory of the 20S proteasome, comparing archaeal and eukaryotic complexes.
• To characterize previously uncharacterized structural features and proteoforms within the 20S proteasome.

Main Methods:

• Utilized native mass spectrometry (MS) tools for a multilevel experimental approach.
• Examined five 20S proteasome complexes from different organisms: *Thermoplasma acidophilum*, *Saccharomyces cerevisiae* (yeast), *Rattus norvegicus* (rat), *Oryctolagus cuniculus* (rabbit), and human (HEK293 cells).
• Employed cryoelectron microscopy single-particle analysis to obtain high-resolution structures and validate MS findings.

Main Results:

• The 20S proteasome has increased in size and stability throughout evolution from archaea to mammals.
• Native MS data revealed high similarity between rat, rabbit, and human 20S proteasomes.
• The yeast proteasome was the largest and most kinetically stable complex; a novel PSMA7 proteoform was identified in rat and rabbit complexes.

Conclusions:

• The developed native MS strategy effectively elucidates structural properties of highly similar protein complexes, including orthologs.
• Evolutionary pressures have driven significant structural diversification and changes in size and stability of the 20S proteasome.
• The findings provide a framework for studying structural variations in related protein assemblies and identified a new proteoform in mammalian proteasomes.