Structural Fluctuations of the Human Proteasome α7 Homo-Tetradecamer Double Ring Imply the Proteasomal α-Ring Assembly Mechanism.

The 20S proteasome, which is composed of layered α and ß heptameric rings, is the core complex of the eukaryotic proteasome involved in proteolysis. The α7 subunit is a component of the α ring, and it self-assembles into a homo-tetradecamer consisting of two layers of α7 heptameric rings. However, the structure of the α7 double ring in solution has not been fully elucidated. We applied cryo-electron microscopy to delineate the structure of the α7 double ring in solution, revealing a structure different from the previously reported crystallographic model. The D7-symmetrical double ring was stacked with a 15° clockwise twist and a separation of 3 Å between the two rings. Two more conformations, dislocated and fully open, were also identified. Our observations suggest that the α7 double-ring structure fluctuates considerably in solution, allowing for the insertion of homologous α subunits, finally converting to the hetero-heptameric α rings in the 20S proteasome.

Mutational and Combinatorial Control of Self-Assembling and Disassembling of Human Proteasome α Subunits.

Eukaryotic proteasomes harbor heteroheptameric α-rings, each composed of seven different but homologous subunits α1-α7, which are correctly assembled via interactions with assembly chaperones. The human proteasome α7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric α6. We comprehensively characterized the oligomeric state of human proteasome α subunits and demonstrated that only the α7 subunit exhibits this unique, self-assembling property and that not only α6 but also α4 can disrupt the α7 double ring. We also demonstrated that mutationally monomerized α7 subunits can interact with the intrinsically monomeric α4 and α6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks.

Two-step process for disassembly mechanism of proteasome α7 homo-tetradecamer by α6 revealed by high-speed atomic force microscopy.

The 20S proteasome is a core particle of the eukaryotic proteasome responsible for proteolysis and is composed of layered α and ß hetero-heptameric rings. The α7 subunit, which is one of components of the α ring, is known to self-assemble into a double-ringed homo-tetradecamer composed of two layers of the α7 heptameric ring. The α7 tetradecamer is known to disassemble upon the addition of α6 subunit, producing a 1:7 hetero-octameric α6-α7 complex. However, the detailed disassembly mechanism remains unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to dissect the disassembly process of the α7 double ring caused by interaction with the α6. HS-AFM movies clearly demonstrated two different modes of interaction in which the α6 monomer initially cracks at the interface between the stacked two α7 single rings and the subsequent intercalation of the α6 monomer in the open pore of the α7 single ring blocks the re-association of the single rings into the double ring. This result provides a mechanistic insight about the disassembly process of non-native homo-oligomers formed by proteasome components which is crucial for the initial process for assembly of 20S proteasome.