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

doi:10.1021/acscentsci.0c00080

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^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

44 Citations (Scopus)

Abstract

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 multilevel 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 Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus 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 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.

Original language	English
Pages (from-to)	573-588
Number of pages	16
Journal	ACS Central Science
Volume	6
Issue number	4
Early online date	10 Apr 2020
DOIs	https://doi.org/10.1021/acscentsci.0c00080
Publication status	Published - 22 Apr 2020

Funding

We thank Dan Tawfik, Weizmann Institute of Science, for helpful discussions. We are grateful for the support of a Starting Grant from the European Research Council (ERC) (Horizon 2020)/ERC Grant Agreement No. 636752, and for an Israel Science Foundation (ISF) Grant 300/17. M.S. is the incumbent of the Aharon and Ephraim Katzir Memorial Professorial Chair. This work was partially supported by a grant from the National Institutes of Health to V.H.W. (NIH P41GM128577). We also thank MS Vision for installing the upgrades on our Synapt G1 instrument.

All Science Journal Classification (ASJC) codes

General Chemistry
General Chemical Engineering

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title = "Comparative Structural Analysis of 20S Proteasome Ortholog Protein Complexes by Native Mass Spectrometry",

abstract = "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 multilevel 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 Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus 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 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.",

author = "Shay Vimer and Gili Ben-Nissan and David Morgenstern and Fanindra Kumar-Deshmukh and Caley Polkinghorn and Quintyn, {Royston S.} and Vasil'ev, {Yury V.} and Beckman, {Joseph S.} and Nadav Elad and Wysocki, {Vicki H.} and Michal Sharon",

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AU - Vimer, Shay

AU - Ben-Nissan, Gili

AU - Morgenstern, David

AU - Kumar-Deshmukh, Fanindra

AU - Polkinghorn, Caley

AU - Quintyn, Royston S.

AU - Vasil'ev, Yury V.

AU - Beckman, Joseph S.

AU - Elad, Nadav

AU - Wysocki, Vicki H.

AU - Sharon, Michal

PY - 2020/4/22

Y1 - 2020/4/22

N2 - 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 multilevel 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 Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus 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 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.

AB - 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 multilevel 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 Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus 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 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.

U2 - 10.1021/acscentsci.0c00080

DO - 10.1021/acscentsci.0c00080

M3 - Article

SN - 2374-7943

VL - 6

SP - 573

EP - 588

JO - ACS Central Science

JF - ACS Central Science

IS - 4

ER -

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

Abstract

Funding

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