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

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.

Cryo-EM of mammalian PA28αβ-iCP immunoproteasome reveals a distinct mechanism of proteasome activation by PA28αβ

“…Taken together, the mammalian PA28αβ may use a mechanism distinct from those of Pf PA28 and Tb PA26 to activate the enzymatic CP. This is in line with a recent Hydrogen–Deuterium eXchange coupled to Mass Spectrometry study indicating that different sets of 20S α-subunit N terminus were destabilized depending on the PA28/20S pair 67 .…”

On the Role of the Immunoproteasome in Protein Homeostasis

“…However, no difference in the binding affinity of the 20S immunoproteasome and standard proteasome to the proteasome activator PA28αβ could be observed [108]. A new study using hydrogendeuterium exchange coupled to mass spectrometry (HDX-MS) describes the existence of allosteric differences between the standard proteasome 20S and the immunoproteasome 20S at the surface of the α-ring triggered from inside the catalytic β-ring [109]. The central pore of the 20S was more flexible in immunoproteasomes.…”

Proteasome complexes experience profound structural and functional rearrangements throughout mammalian spermatogenesis

“…We recently implemented HDX-MS, a technique which 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 (Lesne et al ., 2020). Building on this expertise, we investigated the conformational differences due to the replacement of α4 by α4s.…”

“…Data were collected with MassLynX v.4.1 (Waters Scientific (Manchester, UK) and the robot was controlled via HDX Director v. 1.0.3.9 (LEAP Technologies, Carborro, NC, USA). Each step was optimized to minimize sample loss and work with such a heterogeneous sample, as described previously (Lesne et al ., 2020).…”