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Vimer Ben-Nissan and Sharon Rev Final.pdf (6.23 MB)

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

preprint
submitted on 09.01.2020 and posted on 17.01.2020 by Shay Vimer, Gili Ben Nissan, David Morgenstern, Fanindra Kumar-Deshmukh, Caley Polkinghorn, Royston S. Quintyn, YuryV. Vasli'ev, Joseph S. Beckman, Nadav Elad, Vicki Wysocki, Michal Sharon

Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multi-level experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the T. acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (S. cerevisiae) and mammals (rat - R. norvegicus, rabbit - O. cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native Ms structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution three-dimensional structures, highly resembled that of the human complex. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size, and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

Funding

horizon 2020(Horizon 2020)/ERC Grant Agreement No. 636752

Israel Science Foundation (ISF) Grant 300/17

History

Email Address of Submitting Author

michal.sharon@weizmann.ac.il

Institution

Weizmann Institute of Science

Country

Israel

ORCID For Submitting Author

0000-0003-3933-0595

Declaration of Conflict of Interest

No conflict of interest

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