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

Abstract

The proteasome activator PA28αβ affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28αβ-iCP structure, how PA28αβ regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28αβ-iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28αβ and iCP through XL-MS. Our structures reveal a slight leaning of PA28αβ towards the α3-α4 side of iCP, disturbing the allosteric network of the gatekeeper α2/3/4 subunits, resulting in a partial open iCP gate. We find that the binding and activation mechanism of iCP by PA28αβ is distinct from those of constitutive CP by the homoheptameric TbPA26 or PfPA28. Our study sheds lights on the mechanism of enzymatic activity stimulation of immunoproteasome and suggests that PA28αβ-iCP has experienced profound remodeling during evolution to achieve its current level of function in immune response.

On the Role of the Immunoproteasome in Protein Homeostasis

“…Furthermore, binding of PA28αβ or PA28γ induces conformational changes in the β-rings, the proteasomal active sites, and therefore might modify the 20S products. In addition, the cryo-EM structure of mammalian PA28αβ-immunoproteasome 20S complex suggests that this complex has experienced profound remodelling during evolution to achieve its current level of function in immune response [110]. These data indicate that the binding of regulators to the 20S proteasome allosterically modifies 20S proteasome activity.…”

Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM

“…The protein complex for this previous study was produced by heterologous coexpression of the two subunits, which form a 4α/3β assembly reportedly due to a more thermodynamically stable α-α interface compared to the β-β interface. A more recent cryo-EM study of heterologously expressed human PA28 mixed in vitro with bovine 20S also made a similar conclusion 9 . Interestingly, our study of the endogenous human PA28-20S proteasome revealed a different stoichiometry of PA28.…”

“…The other six unique binding pockets are well engaged with the corresponding C termini of the PA28 3α/4β assembly. On the other hand, the PA28 4α/3β assembly derived from heterologous coexpression showed only partial association with native 20S when assembled in vitro 9 . These results suggest that PA28 in a 4α/3β arrangement has reduced binding affinity to 20S compared to a 3α/4β shows PA28 associating tightly with 20S, resulting in well-defined density for the entire complex.…”

“…While the proteasome has been a topic of extensive studies primarily centering on the bacterial homologues, the study of the mammalian proteasome remains challenging due to its structural complexity. Previous studies have utilized complex strategies to capture distinct human proteasomal complexes [9][10][11][12] , but a general strategy to analyze different proteasomal complexes from the same system and at the same time is not available. Here, we demonstrate the ability to visualize and study multiple proteasomal complexes simultaneously by single particle cryo-EM.…”

PA28γ–20S proteasome is a proteolytic complex committed to degrade unfolded proteins

“…Interestingly, in this case PA28 was found to bind 20S asymmetrically, strongly engaging subunits on only one side of the core particle [9]. However, strong evidence indicating that PA28 may also act through long distance allosteric modifications of proteasomal proteolytic sites has been recently reported [10][11][12].…”