Structural biology of the proteasome

Abstract

The proteasome refers to a collection of complexes centered on the 20S proteasome core particle (20S CP), a complex of 28 subunits that houses proteolytic sites in its hollow interior. Proteasomes are found in eukaryotes, archaea, and some eubacteria, and their activity is critical for many cellular pathways. Important recent advances include inhibitor binding studies and the structure of the immunoproteasome, whose specificity is altered by the incorporation of inducible catalytic subunits. The inherent repression of the 20S CP is relieved by the ATP-independent activators 11S and Blm10/PA200, whose structures reveal principles of proteasome mechanism. The structure of the ATP-dependent 19S regulatory particle, which mediates degradation of polyubiquitylated proteins, is being revealed by a combination of crystal or NMR structures of individual subunits and electron microscopy reconstruction of the intact complex. Other recent structural advances inform us about mechanisms of assembly and the role of conformational changes in the functional cycle.

Figure 1. 20S proteasome core particle (20S CP)

(a) Side view of the archaeal T. acidophilum 20S CP (51) (pdb code 1pma). End rings comprise seven identical α subunits (brown) and the two middle rings comprise seven identical β subunits (dark blue). (b) Side view of the eukaryotic S. cerevisiae 20S CP (24) (pdb code 1ryp). Each of the seven different α subunits and seven different β subunits occupies a unique position within their respective rings. The whole structure has two-fold symmetry relating the top and bottom halves of the structure to each other, with the 2-fold axis in the horizontal plane, a little to the right of center in this view. (c) Cutaway view showing internal features. The S. cerevisiae 20S CP is shown in ribbon representation with just eight subunits displayed in order to reveal the hollow interior. Labeled features include residues that contribute to the asymmetric closed gate structure, loops that contribute to the α annulus, and the active sites of β1 and β5 in the lower β ring (only the β1, β2, and β5 subunits have active sites in eukaryotic proteasomes). (d) Conformational changes at the activate site of M. tuberculosis 20S CP that are induced upon binding of inhibitor suggest the possibility of developing a specific therapeutic (49). The loop connecting S4 and H1 of the β subunit moves from the unbound conformation (white, pdb code 2fhg) to cover OXZ, the inhibitor oxazolidin-2-one ring on Thr1 in the stabilized complex (purple, pdb code 3h6f). (e) Comparison of mouse liver constitutive and inducible β5 S1 binding pocket (30). Met45 adopts the sky blue conformation (pdb code 3unf) when bound to the PR-957 inhibitor (pink), which binds with a large hydrophobic group in the S1 pocket. Met45 also adopts this conformation in the unbound immunoproteasome but adopts the tan conformation in the unbound constitutive proteasome (pdb code 3une). This requirement for repositioning Met45 explains why immunoproteasomes prefer to cleave substrate after large hydrophobic side chains.

Figure 2. ATP-independent activators

(a) Top - crystal structure of the T. brucei PA26 heptamer (yellow) in complex with S. cerevisiae 20S CP (18) (pdb code 1z7q). Middle - side view of PA26 ribbon representation with each of the seven identical subunits in a different color. Bottom - PA26 top view. Loops from an insertion in helix 3 project into the middle of the channel where they would impede transit of a potential substrate. (b) Top - crystal structure of the S. cerevisiae Blm10-20S CP complex (68) (pdb code 1vsy). Middle - side view of Blm10, rainbow colored from N-terminus (blue) to C-terminus (red). Bottom - top view. (c) Top - top surface of S. cerevisiae 20S CP in the unbound closed conformation. Middle - closer view (corresponding to frame of top panel) showing the open conformation induced by PA26 and the four ordered PA26 C-termini visible in this structure. Bottom -top surface of S. cerevisiae 20S CP from the Blm10 complex structure. The gate appears open, although not so extensively as with PA26, and the space is largely filled with disordered residues, which are indicated as white ribbons.

Figure 3. 19S regulatory particle/26S proteasome

Top left, boxed, two views of a cartoon depiction of the 26S proteasome EM structures showing the 20S CP (tan), base (cyan), and lid (purple). A charge density map of the S. cerevisiae 26S proteasome reconstruction (43) is shown centrally. Atomic models for individual protein subunits whose structures are known at atomic resolution have been positioned following the analyses of references (43) and (3), and are shown around the periphery in an expanded view. Also included are Rad23, Ubp6/USP14, and the C-terminal domain of human Rpn13, which are not part of the reconstructed complex but illustrate how additional structural components contribute to proteasome function. See box for details.

Figure 4. Structures of proteasome chaperones

(a) Pba1-Pba2 (orange and blue) structure from (83) (pdb code 4g4s). Side and top views are shown of the complex with the 20S CP. The contacts seen in this structure are presumably maintained from the earliest stages of α-ring assembly to maturation of the 20S CP. (b) Pba3-Pba4 (shades of blue) structure from (100) (pdb code 2z5c). Side and bottom views are shown of this complex with α5, with the other α subunits modeled in white based on their structure in the mature 20S CP. This structure explains why Pba3-Pba4 are lost as β subunits are added to the assembling 20S CP. (c) Structures of 19S RP chaperones. Rpn14 (36) (pdb code 3acp), Hsm3 complex with C-terminal domain of Rpt1 (1) (pdb code 4a3v), and Nas6/gankyrin complex with the C-terminal domain of Rpt3 (54) (pdb code 2dzn). (d) Side and bottom model of Hsm3 and Nas6 docked onto the Rpt hexamer model. Substantial steric clashes would occur with the 20S CP (not shown) in the 26S proteasome, and minor steric clashes are suggested with Rpt subunits, consistent with the mechanisms that the 19S RP chaperones modulate interactions between ATPase subcomplexes and with the 20S CP. The C-terminal domains of Rpt5 and Rpt6 that bind Nas2 and Rpn14 are colored green and pink, respectively.