Abstract
The 20S core particle of the eukaryotic proteasome is composed of two α- and two β-rings, each of which is a hetero-heptamer composed of seven homologous but distinct subunits. Although formation of the eukaryotic proteasome is a highly ordered process assisted by assembly chaperones, α7, an α-ring component, has the unique property of self-assembling into a homo-tetradecamer. We used biophysical methods to characterize the oligomeric states of this proteasome subunit and its interaction with α6, which makes direct contacts with α7 in the proteasome α-ring. We determined a crystal structure of the α7 tetradecamer, which has a double-ring structure. Sedimentation velocity analytical ultracentrifugation and mass spectrometric analysis under non-denaturing conditions revealed that α7 exclusively exists as homo-tetradecamer in solution and that its double-ring structure is disassembled upon the addition of α6, resulting in a 1:7 hetero-octameric α6–α7 complex. Our findings suggest that proteasome formation involves the disassembly of non-native oligomers, which are assembly intermediates.
Highlights
Is lower than that in the MS method
Recent sophisticated SV-AUC analyses have provided us with information regarding the structures of proteins and their complexes in solution, based on comparisons of experimentally estimated hydrodynamic parameters, e.g., sedimentation coefficient and diffusion constant, with those computed from their three-dimensional structure models[12]
We determine a crystal structure of the human α 7 homo-tetradecamer and apply the complimentary MS and SV-AUC methods to investigate the oligomeric states of the proteasome α subunits, focusing on α 7 and α 6, its neighbor in the correctly arranged α -ring
Summary
The SV-AUC data showed that the α 7 subunit exclusively exists as a single species with a sedimentation coefficient of 14.2 S (Fig. 2a), which is in excellent agreement with that estimated from the crystal structure (14.4 S), confirming the double-ring structure of this complex in solution. The molecular mass determined for this complex was 227,843 ± 87 Da, which corresponds to a 7:1 complex of α 7 and α 6 (with a calculated mass of 227,394 Da) All these data indicate that α 6 interacts with α 7, disassembling one mole of the tetradecameric double-ring of α 7 and thereby give rise to two moles of the 1:7 hetero-octameric α 6–α 7 complex. Our findings will provide new clues for drug discovery targeting the assembly/disassembly intermediates generated during proteasome formation
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