Cryo-EM of mammalian PA28 alpha beta-iCP immunoproteasome reveals a distinct mechanism of proteasome activation by PA28 alpha beta

The proteasome activator PA28 alpha beta affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28 alpha beta -iCP structure, how PA28 alpha beta regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28 alpha beta -iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28 alpha beta and iCP through XL-MS. Our structures reveal a slight leaning of PA28 alpha beta towards the alpha 3-alpha 4 side of iCP, disturbing the allosteric network of the gatekeeper alpha 2/3/4 subunits, resulting in a partial open iCP gate. We find that the binding and activation mechanism of iCP by PA28 alpha beta 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 PA28 alpha beta -iCP has experienced profound remodeling during evolution to achieve its current level of function in immune response. The proteasome activator PA28 alpha beta affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). Cryo-EM structures of the mammalian PA28 alpha beta -iCP immunoproteasome and free iCP, combined with cross-linking data, reveal the complex architecture and suggest a distinct immunoproteasome activation mechanism.