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

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 PA28 alpha beta or PA28 gamma 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 alpha -ring triggered from inside the catalytic beta -ring. Additionally, binding of the PA28 regulators to the 20S proteasomes modify solvent accessibility due to conformational changes of the beta -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.