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

• Published in: Nature communications 2020-12, Vol.11 (1), p.6140-15, Article 6140
• Main Authors: Lesne, Jean, Locard-Paulet, Marie, Parra, Julien, Zivković, Dušan, Menneteau, Thomas, Bousquet, Marie-Pierre, Burlet-Schiltz, Odile, Marcoux, Julien
• Format: Article
• Language: English
• Subjects: 140/58; 631/1647/296; 631/45/474/2085; 631/535; 631/57/2272/2276; Accessibility; Allosteric properties; Allosteric Regulation; Analytical chemistry; Autoantigens - chemistry; Autoantigens - genetics; Autoantigens - metabolism; Binding; Biochemistry, Molecular Biology; Catalytic subunits; Chemical Sciences; Deuterium; Humanities and Social Sciences; Humans; Hydrogen; Hydrogen-deuterium exchange; Immune system; Immunogenicity; Life Sciences; Ligands; Mass Spectrometry; Mass spectroscopy; multidisciplinary; Muscle Proteins - chemistry; Muscle Proteins - genetics; Muscle Proteins - metabolism; NMR; Nuclear magnetic resonance; Peptides; Proteasome Endopeptidase Complex - chemistry; Proteasome Endopeptidase Complex - genetics; Proteasome Endopeptidase Complex - metabolism; Proteasomes; Protein Binding; Proteins; Regulation; Regulators; Science; Science (multidisciplinary); Scientific imaging; Solvents; Statistical analysis; Structural Biology
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-19934-z

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. Immune cells express immunoproteasomes (i20S), which bind to specialized regulators, contain different catalytic subunits and generate immunogenic peptides. HDX-MS—based assessment of the differences between the conformational dynamics of standard and i20s reveals specific, allosteric changes in i20S and upon regulator binding.