Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry
2020
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.
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