Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependent inter-ring crosstalks

Jean Lesne,Odile Burlet-Schiltz,Thomas Menneteau,Julien Marcoux,Julien Parra,Dušan Zivković,Marie Locard-Paulet,Marie-Pierre Bousquet

doi:10.1038/s41467-020-19934-z

Abstract

Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS) is now common practice in structural biology. However, it is most of the time applied to rather small oligomeric complexes. Here, we report on the use of HDX-MS to investigate conformational differences between the human standard 20S (std20S) and immuno 20S (i20s) proteasomes alone or in complex with PA28αβ or PA28γ activators. Their solvent accessibility is analyzed through a dedicated bioinformatic pipeline including stringent statistical analysis and 3D visualization. These data confirm the existence of allosteric differences between the std20S and i20S at the surface of the α-ring triggered from inside the catalytic β-ring. Additionally, binding of the PA28 regulators to the 20S proteasomes modify solvent accessibility due to conformational changes of the β-rings. This work is not only a proof-of-concept that HDX-MS can be used to get structural insights on large multi-protein complexes in solution, it also demonstrates that the binding of the std20S or i20S subtype to any of its PA28 activator triggers allosteric changes that are specific to this 20S/PA28 pair.

Highlights

Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-mass spectrometry (MS)) is common practice in structural biology
The catalytic activation observed upon PA28γ/standard 20S (std20S) binding may be driven by changes occurring further away from the active sites. This structural dataset on the human i20S compared to the std20S alone or bound to both PA28 regulators rationalizes from a mechanistic point of view previous observations that replacement49, modification34 or ligand binding49,55 to the catalytic subunits can allosterically modify the 20S core particle structure and alter its binding to potential Proteasome Interacting Proteins (PIPs)
It is not theoretically limited by the complex size since the proteins are digested into peptides after deuteration: it can be applied to very small proteins or very large complexes

Summary

Introduction

Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS) is common practice in structural biology. We report on the use of HDX-MS to investigate conformational differences between the human standard 20S (std20S) and immuno 20S (i20s) proteasomes alone or in complex with PA28αβ or PA28γ activators. Their solvent accessibility is analyzed through a dedicated bioinformatic pipeline including stringent statistical analysis and 3D visualization. Besides its subunit composition and association with regulators and/or PIPs, the proteasome is subjected to a wide variety of posttranslational modifications (PTMs) affecting its subunits activity17–19 The combination of these three levels of regulation (catalytic subunits, regulators, PTMs) implies a wide proteasome subtype heterogeneity, which probably results in specialized functions that can adapt protein degradation pathways to changing conditions in the cell. One of today’s main challenges is to understand how different proteasome subtypes influence its proteolytic activity and the cell function

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Results

Conclusion