Comparative Structural Analysis of 20S Proteasome Ortholog Protein Complexes by Native Mass Spectrometry
Shay Vimer, Gili Ben‐Nissan, David Morgenstern, Fanindra Kumar Deshmukh, Caley Polkinghorn, Royston S. Quintyn, Yury V. Vasil’ev, Joseph S. Beckman, Nadav Elad, Vicki H. Wysocki, Michal Sharon
Abstract
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 cryoelectron 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.