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

Abstract

Here, we used for the first time Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (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 was analyzed through a dedicated bioinformatic pipeline including stringent statistical analysis and 3D visualization. These data confirmed 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 modified 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.

Proteasome complexes experience profound structural and functional rearrangements throughout mammalian spermatogenesis

“…HDX-MS and MD Simulations. HDX-MS experiments were performed on a Synapt-G2Si (Waters Scientific) coupled to a Twin HTS PAL dispensing and labeling robot (Trajan Scientific) via a NanoAcquity system with HDX technology (Waters Scientific), as described previously (47). More details, as well as MD simulation workflow, can be found in SI Appendix, SI Experimental Procedures.…”

“…Singularity. We recently implemented HDX-MS, a technique that reports on solvent accessibility and/or flexibility of the backbone amide protons, on the c20S and i20S proteasome complexes, in order to decipher their structural rearrangements upon replacement of their catalytic subunits and binding to PA28 regulators (47). Building on this expertise, we investigated the conformational differences due to the replacement of α4 by α4s.…”

Effector-mediated subversion of proteasome activator (PA)28αβ enhances host defense againstLegionella pneumophilaunder inflammatory and oxidative stress conditions

“…These data together with our observation that the LegC4 phenotype is recapitulated in PA28ab-deficient macrophages have led us to speculate that LegC4 binding prevents PA28-CP complex formation. While the function of AcPA28 has not been described, structural studies have shown that mammalian PA28a and evolutionary distant PA28s are similar and all associate with proteolytic proteasome core particles, suggesting that it likely also functions as a proteasome regulator (33,34). The constitutive 26S proteasome system is central to L. pneumophila's virulence strategy and its integrity may be preserved by sequestering PA28 from CPs.…”

“…evolutionary distant PA28s are similar and all associate with proteolytic proteasome core particles, suggesting that it likely also functions as a proteasome regulator (Lesne et al, 2020; Xie et al, 2019). The constitutive 26S proteasome system is central to L. pneumophila ’s virulence strategy and its integrity may be preserved by sequestering PA28 from CPs.…”

Allosteric regulation of the proteasome’s catalytic sites by the proteasome activator PA28γ /REGγ

“…In support of this notion, a recent study by Lesne et. al used Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS) to determine that PA28αβ and PA28γ adopt different conformations when binding the proteasome, as the activation loops and helix 1 of PA28αβ were heavily protected from HDX solvent exchange when bound to the proteasome compared to PA28γ 42 . Our structure of the human PA28γ-20S complex reveal that PA28γ adopts a quaternary topology similar to other structurally characterized 11S regulators (i.e., PA26, PA28 Pf , and PA28αβ).…”