Disassembly of the self-assembled, double-ring structure of proteasome α7 homo-tetradecamer by α6

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.

Scientific RepoRts | 5:18167 | DOI: 10.1038/srep18167 is lower than that in the MS method. In addition, 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 .
Here 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.

Results & Discussion
Structure determination of the α7 homo-tetradecamer. First, we determined the crystal structure of the human α 7 tetradecamer at 3.75-Å resolution. The α 7 tetradecamer exhibited an hourglass double-ring shape (Fig. 1a,b), which is consistent with the previously reported negative staining electron micrographs 9 and small-angle neutron scattering (SANS) data 10 . The two α rings interact with each other mainly through two α helices that are involved in β -subunit interaction in the 20S proteasome 5,13 . The conformations of the individual protomers and their quaternary arrangement in each ring of the human α 7 tetradecamer are essentially identical with those observed in the homo-heptameric α -ring in archaeal proteasomes 14 and the hetero-heptameric α -ring in mammalian proteasomes (Fig. 1c,d) 5,13 . Oligomeric states of proteasome α7 and α6 subunits. To characterize the oligomeric state of the α 7 subunits in solution, we performed MS and SV-AUC analyses. 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 mass spectrum of α 7 under non-denaturing conditions exhibited ion series indicating Figure 1. Crystal structure of the human α7 homo-tetradecamer. Ribbon models of the single and double α 7 rings derived from the human α 7 tetradecamer are shown in (a,b), respectively. Ribbon models of the α 1-7 ring and the α 1-7-β 1-7 ring (half-proteasome) derived from the human 20S proteasome (PDB code 4R3O) are shown in (c,d), respectively. The left and right structures are related by a rotation of 90° around a horizontal axis. The α subunits are colored as follows: α 1 (blue), α 2 (green), α 3 (magenta), α 4 (forest green), α 5 (orange), α 6 (cyan), and α 7 (red). a complex with a molecular mass of 405,879 ± 81 Da (Fig. 2b). This confirms the homo-tetradecameric structure of this subunit (with a calculated mass of 28,284 Da, based on its amino acid sequence). Therefore, these results are consistent with the present crystal structure and the previously reported electron microscopy and solution scattering observations 9,10 .
Furthermore, we examined the oligomeric state of the human proteasome α 6 subunit, which makes direct contact with the α 7 subunit in the proteasomal hetero-heptameric α -ring. The SV-AUC analysis showed that a majority of α 6 exists as a monomer (2.6 S peak), while indicating that this subunit has a tendency to aggregate (Fig. 3a). The mass spectrum of α 6 under non-denaturing conditions exhibited one major and one minor ion series, corresponding to the molecular masses of the monomer (29,486 ± 0 Da) and dimer (59,003 ± 11 Da), respectively (Fig. 3b).
To address the structural basis of the distinct oligomeric properties between α 6 and α 7, we made a hypothetical model of two α 6 subunits brought into juxtaposition based on the homo-tetradecameric structure of α 7 ( Supplementary Fig. S1). Although a detailed comparison of the inter-subunit interaction modes was difficult because of the low resolution of the present crystal structure, the model clearly shows that α 6 makes steric hindrances at the inter-subunit interface, thereby explaining why α 6 is not able to form the homo-heptameric ring as α 7 does.
Disassembly of the α7 double ring upon the addition of α6. Further, we attempted to characterize the oligomeric states of a mixture of α 7 and α 6 using SV-AUC and MS. The SV-AUC data indicated that the α 7 homo-tetradecamer was less populated in the presence of α 6, with the appearance of a smaller complex with a sedimentation coefficient of 10.2 S (Fig. 4a). In the MS analysis, the ion peak intensities of the α 7 homo-tetradecamer were attenuated on titration with α 6, with concomitant appearance of a new peak series (Fig. 4b). 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. The mass spectrometric data showed that the α 7 homo-tetradecamer was residual even in the presence of excess amount of α 6, indicating that α 7 was under equilibrium between the homo-tetradecameric state and the hetero-octameric form with α 6 under the experimental condition. The mass spectra acquired at 60 min (upper) and 115 min (lower) after mixing of α 6 and α 7 were virtually identical in terms of the population of the α 7 homo-tetradecamer and the α 6-α 7 hetero-octamer, implying that the system reached equilibrium within 60 min ( Supplementary Fig. S2). The sedimentation coefficient of a single homo-heptameric α 7 ring was calculated to be 8.9 S from the present crystal structure, suggesting that the 10.2 S hetero-octamer had a ring-shaped structure, although the position of α 6 was uncertain.
Two alternative models for the disassembly of homo-heptameric α 7 could be hypothesized. In one model, α 7 exists in equilibrium between the double-and single-ring forms and α 6 traps and stabilizes the single-ring species. However, our previous SANS data indicated that no detectable ring exchange occurred for 14 h after mixing deuterated and non-deuterated α 7 homo-tetradecamers, indicating that the two heptameric rings are tightly associated with each other in the absence of the α 6 subunit 15 . The other model is that α 6 binds the α 7 homo-tetradecamer and induces some type of allosteric conformational change at the ring-ring interface, resulting in the disassembly of the double-ring structure. Considering the stability of the α 7 double-ring form in the absence of α 6, the latter model is more plausible, although an α 6-bound homo-tetradecameric species of α 7 could be detected in neither the SV-AUC profiles nor the mass spectra. Further kinetic and structural studies are necessary to identify the disassembly mechanism and quaternary structure of the α 6-α 7 hetero-octamer.   In summary, the present study demonstrates that the proteasome α 6 subunit acts as a breaker of the α 7 double-ring structure. Our findings suggest that proteasome formation involves disassembly processes of non-native oligomeric forms of proteasome subunits as assembly intermediates. The proteasome assembly pathway is a potential target for anticancer drug development [16][17][18] . Therefore, our findings will provide new clues for drug discovery targeting the assembly/disassembly intermediates generated during proteasome formation.

Methods
Protein expression and purification. Human proteasome α 6 short isoform and α 7 subunits were expressed using Escherichia coli strain BL21-CodonPlus and purified as described previously 10,15 . For the preparation of recombinant proteins, the cells were grown in Luria-Bertani medium. After sonication and centrifugation, cell lysates were subjected to anion-exchange chromatography (DEAE Sepharose Fast Flow, GE Healthcare). The proteins were further purified using an anion-exchange HPLC column (RESOURCE Q, GE Healthcare) and then with a gel-filtration HPLC column (HiLoad 26/60 Superdex 200 pg, GE Healthcare). Crystallization, X-ray data collection, and structure determination. Native and selenomethione (SeMet)-substituted human α 7 (10 mg/mL) was dissolved in 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl. Crystals were grown in a buffer containing 24% PEG400, 100 mM Tris-HCl (pH 7.5), and 0.2 M magnesium chloride. These mixtures were incubated at 20 °C for approximately 1 week. The crystals were cryoprotected with the reservoir solution and flash-cooled in liquid nitrogen. The native and SeMet-substituted crystals belonged to space group P4 3 2 1 2, with one α 7 tetradecamer per asymmetric unit. They diffracted at resolutions up to 3.75-and 4.20-Å resolution, respectively. Diffraction data were scaled and integrated using HKL2000 19 .
The 3.75-Å resolution crystal structure of the α 7 tetradecamer was solved by the molecular replacement method using the program MOLREP 20 with a single α -ring of archaeal proteasome (Protein Data Bank code 1J2P) 14 as the search model. Subsequently, fourteen bovine α 7 subunit copies (PDB code 1IRU) 5 were superimposed and then replaced with the archaeal α subunits. Model fitting to the electron density maps was performed using COOT 21 , in conjunction with the SeMet anomalous data. REFMAC5 22 was used for the crystal structure refinement, and the stereochemical quality of the final model was validated using PROCHECK 23 . The crystal parameters and refinement statistics are summarized in Supplementary Table S1. The molecular graphics were prepared using PyMOL (http://www.pymol.org/).

Sedimentation velocity analytical ultracentrifugation. Sedimentation analytical ultracentrifugation
experiments were performed in 150 mM potassium phosphate (pH 7.4), using a ProteomeLab XL-I Analytical Ultracentrifuge equipped with four-hole An60 Ti rotor (Beckman Coulter). Solutions loaded in epon or aluminum centerpieces (Beckman Coulter) were run at 25,000 rpm for α 7 and α 7 + α 6, and at 55,000 rpm for α 6. Data were collected using an absorbance optical system at wavelengths where absorbance values were between 0.8 and 1.2. Data were analyzed using the continuous c (s) distribution model in the program SEDFIT (version 14.4d). The partial specific volume of α 6 and α 7, buffer viscosity, and buffer density, calculated using the program SEDNTERP 1.09, were 0.73035 cm 3 /g, 0.71588 cm 3 /g, 1.013 cP, and 1.0009 g/mL, respectively. Hydrodynamic parameters were calculated from the crystallographic data with a program SOMO (SOlution MOdeller) equipped with the UltraScan 3 12 .