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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Mol Biol. Author manuscript; available in PMC Mar 13, 2010.
Published in final edited form as:
PMCID: PMC2671960
NIHMSID: NIHMS102904

Electron microscopic evidence in support of alpha-solenoid models of proteasomal subunits, Rpn1 and Rpn2

Summary

Rpn1 (109 kDa) and Rpn2 (104 kDa) are components of the 19S regulatory complex of the proteasome. The central portions of both proteins are predicted to have toroidal α-solenoid folds composed of 9–11 PC (proteasome/cyclosome) repeats, each ~ 40 residues long and containing two α-helices and turns (A.V. Kajava, J. Biol. Chem. 277, 49791–8, 2002). To evaluate this prediction, we examined the full-length yeast proteins and truncated (PC) versions thereof consisting only of the repeat-containing regions by gel filtration, circular dichroism spectroscopy, and negative staining electron microscopy. All four proteins are monomeric in solution and highly α-helical – particularly, the truncated ones. The EM data were analyzed by image classification and averaging techniques. The preponderant projections, in each case, show near-annular molecules, 6–7 nm in diameter. Comparison of the full-length with the truncated proteins showed molecules similar in size and shape, indicating that their terminal regions are flexible and thus smeared to invisibility in the averaged images. We tested the toroidal model further by calculating resolution-limited projections and comparing them with the EM images. The results support the α-solenoid model, except that they indicate that the repeats are organized not as symmetrical circular toroids but in less regular horseshoe-like structures.

Keywords: negative staining electron microscopy, image classification, circular dichroism, alpha-solenoids

Energy-dependent proteolysis plays a key role in prokaryotic and eukaryotic cells by restricting the availability of certain short-lived regulatory proteins, ensuring the proper stoichiometry for multi-protein complexes, and eliminating misfolded, mislocalized or damaged proteins 1; 2. In eukaryotes, the proteasome is responsible for the degradation of ubiquitin-tagged substrates. The fully assembled 26S particle comprises two subcomplexes: the 20S proteolytic chamber formed of four heptameric rings; and the end-mounted 19S complex that regulates substrate processing. The 19S complex may be subdivided into a base and a lid 3; 4. The distally positioned lid is a complex of eight subunits implicated in deubiquitination and the initial steps of substrate processing 5; 6. The base stacks on to the apical surface of the 20S barrel and consists of a ring of six AAA+ ATPase subunits, Rpt1–6, and four other proteins - Rpn1, Rpn2, Rpn10 and Rpn13 7. Rpn10 and Rpn13 have ubiquitin-binding domains 8; 9 and Rpn13 can also bind the de-ubiquitinating enzyme, Uch37 10. The remaining base components, Rpn1 and Rpn2, are the largest proteasomal subunits and have been reported to interact with several other subunits 8; 9; 11; 12; 13 and auxiliary factors 14; 15 and with each other 16.

Whereas the structure of the 20S core particle is known to high resolution 17, structural information on the 19S regulatory particle remains limited. 3D reconstructions from electron microscopy 18; 19 and tomography 20 have provided an overall frame of reference, but there has been little information on the placement of components other than the AAA+ ring or on the detailed structures of individual subunits, apart from Rpn13 8; 21.

Based on bioinformatic analysis, it was deduced that Rpn1 and Rpn2 both have central regions containing 9–11 tandem pseudo-repeats of an α-helical motif 22. Repeats of this kinds are quite widespread and several subfamilies have been identified, including the so-called HEAT and ARM repeats 23. The Rpn1 and Rpn2 sequence repeats are of a kind associated with proteins of the Proteasome/Cyclosome family 24 and thus are called PC repeats. In tertiary structures, these repeats stack, giving folds that are generically called α-solenoids. Several crystal structures have been determined and the solenoids found to vary in shape from highly curved to nearly straight 25. Based on the periodic occurrence of residues with small side chains, it was anticipated that the α-solenoids of Rpn1 and Rpn2 would be highly curved, and toroidal models were proposed for both 22. In the present study, we aimed to test these predictions by circular dichroism spectroscopy and negative staining electron microscopy as applied to both the full-length proteins and truncated forms, restricted to the PC-containing regions.

Rpn1 and Rpn2, truncated to their PC regions, are alpha-helical monomers

The full-length and truncated forms of Rpn1 and Rpn2 were expressed in E. coli and purified (see Fig. 1 legend). In gel filtration, all four proteins elute at positions corresponding to their monomeric molecular weights (Fig. 1). These data are consistent with prior evidence from analytic ultracentrifugation that the full-length proteins are monomeric in solution 16, and establish that the truncated proteins share this property.

Fig. 1
Expression and purification of Rpn1 and Rpn2 and truncated versions thereof. (a) SDS-PAGE (10% gel) of purified recombinant proteins stained with Coomassie Blue: Rpn1 (lane 1), Rpn1PC (lane 2), Rpn2 (lane 3), and Rpn2PC (lane 4). (b) Gel-filtration chromatography ...

We investigated their secondary structures by circular dichroism spectroscopy. The resulting spectra (Fig. 2), which are characteristic of α-helical proteins 26, were used to estimate their secondary structure contents. In both cases, the truncated proteins have a higher helical content than the full-length proteins (~ 55% vs ~ 35%) and a negligible content of β-sheets. When allowance is made for the presence of inter-helical turns and a reasonable margin of experimental error, these data are consistent with the PC proteins being almost entirely α-solenoidal. This analysis also assigns a substantial amount of non-regular secondary structure to the full-length proteins.

Fig. 2
Circular dichroism spectra in the far-UV range (190–260 nm) of recombinant Rpn1, Rpn1PC, Rpn2, and Rpn2PC. Prior to CD analysis, each protein was dialyzed (three times, 100 vol. of sample) against 25mM Borate buffer (pH 7.4). Data were recorded ...

Visualized by negative staining, Rpn1 and Rpn2 have central stain-accumulating regions

The four proteins were examined by negative staining EM. In all cases, the predominant species observed was small globular particles ( Supp. Fig. 1a–d), many with a stain accumulation at their center. The micrographs were digitized and the resulting data analyzed to distinguish the various classes of particles, and then averaged within each class to improve the signal-to-noise ratio and thus the interpretability of the images. The 10 classes distinguished for full-length Rpn2 are shown in the top row of Figure 3, ordered according to shape similarity. We assign the classes to four groups. Five of the classes (group 1), accounting for ~ 58% of the data, present tetrads of stain-excluding densities, 6 to 7 nm in total diameter, surrounding a central stain accumulation, ~ 1.5 to 2 nm across. The group 1 classes differ in relatively subtle features, such as how clearly densities are resolved from their neighbors or the extent to which their distribution departs from a square. In the leftmost class, the top right density appears to extend out through the surrounding stain layer. The four group 2 classes (~ 30% of the data) show a generally similar molecule, i.e. of about the same size and also with a stain-accumulating center, but with the densities less clearly resolved. The single group 3 class depicts a pair of densities whose length matches the diameter of the other classes. It is plausibly explained as a side-view, in which case its central transverse stripe indicates that the stain-accumulating features at the centers of the other classes represent a hole passing through the molecule.

Fig. 3
Negative stain electron microscopy. 2D class averages are shown for Rpn2 (row 1), Rpn2PC (row 2), Rpn 1 (row 3) and Rpn1PC (row 4). Microscopy. The purified proteins were brought to final concentrations of 7.5 to 15 μg/mL in 50 mM HEPES and 150 ...

Analysis of full-length Rpn1 (Fig. 3, row 3) had a similar outcome. Here, the large majority of the data were assigned to 11 classes and again, tetrad-presenting classes predominated (seven of ten classes, with ~ 71% of the data, are in group 1). Compared with Rpn2, more of these images show extensions of the top right density, to varying lengths, in classes 1 to 4 of group 1.

The PC repeat regions form the central cores of Rpn1 and Rpn2

The class averages obtained for the truncated proteins are similar in shape and size to those of the full-length proteins, despite missing nearly half their masses (Fig. 3, rows 2 and 4). Moreover, they can be matched fairly consistently with the group 1 and 2 images defined for the full-length proteins. The truncated proteins also having an abundance of tetrad-presenting particles (40% and 50% of the total data for Rpn2PC and Rpn1PC). Both truncated proteins also have a single triad-presenting class (group 4, ~ 8% of both data sets). However, no class average of either truncated protein showed an “extending arm” domain, as seen for both of the full-length proteins (Rpn2; group 1, class 1 and Rpn1; group1, classes 1 – 4). These observations clearly assign the tetrads of density to the central PC repeat-containing regions of Rpn1 and Rpn2. It is noteworthy that neither truncated protein shows the “extending arm” seen in class averages of the full-length proteins. This observation leads us to infer that the arm is contributed by a terminal region. On the other hand, the variability of this feature implies that the terminal regions of the full-length proteins are relatively flexible.

Modeling the PC repeat regions of Rpn1 and Rpn2 as curved α-solenoids

The PC repeat regions of both proteins have been predicted to form toroidal α-solenoids 22. The size of the molecules that we observe (Fig. 3) and their hollow centers are consistent with this prediction. To test it further, we calculated a set of projections corresponding to different views of this model and of other related models (Fig. 4a), and band-limited them to 2.5 nm resolution (Fig. 4b, row 2–5) for comparison with the EM data (Fig. 4b, row 1). The diameters of the EM class averages are slightly more consistent with a 11-repeat model (6.0 nm vs. 5.5 nm for the 9-repeat model, Supp. Fig. 2), although they do not rule out the latter model when the possibility of molecules spreading laterally when dried in negative stain and the limited resolution are borne in mind.

Fig. 4
Modeling of Rpn2 PC and comparison of the resulting structures with the EM class averages. (a) Ribbon representations of four models. Model 1: a symmetric 11-repeat toroid; Models 2 and 3: manually distorted 11-repeat toroid; Model 4: predicted PC repeat ...

The most pronounced feature of the EM class averages is the resolution of the ring into four (or three) densities of approximately equal size. This feature is not reproduced in any viewing direction of a symmetrical circular toroid (Fig. 4b, row 2). However, it may be produced by distorting the toroid in any of several ways. Models 2 and 3 in Figure 4a were generated by distorting the 11-repeat circular planar toroid and slightly opening the ring at the gap where the N and C-termini of the PC repeat region meet. We also generated another 11-repeat model for Rpn2PC, using structure of the nuclear transport protein, karyopherin β2 - a known α-solenoid 27 - as template (model 4). Near-axial projections of models 2 to 4 have an overall horseshoe shape and, at lower resolution, display tetrads of density (Fig. 4b, row 3–5), in agreement with the EM class averages (Fig. 4b, row 1).

We conclude that this analysis supports the prediction that the PC repeat portions of Rpn2 and Rpn1 (Supp. Fig. 3) form monomeric α-solenoids with highly curved folds, although they appear to be less regular than in the simple symmetrical model 22. The slightly opened model that we favor over a closed one (model 3), allows the N- and C-terminal regions to extend away from the PC repeat regions.

Implications for 19S structure

In a previous study 16, isolated molecules of full-length Rpn1 and Rpn2 imaged by AFM were reported to have a thickness and annular appearance consistent with the toroidal α-solenoid model 22. AFM affords precise height measurements but tip convolution effects compromize its lateral resolution. Nevertheless, our class-averaged EM images and modeling experiments support and extend this conclusion. In particular, the close resemblance between the images of the full-length and the truncated proteins assigns the α-solenoid fold to the PC repeat-containing portions of these molecules. Moreover, the “extending arm” seen only with full-length proteins, particularly Rpn1 (Fig. 3, row 3), is likely to represent part of their terminal regions. As the C-terminal part of Rpn1 is rather short (117 residues), its “extending arm” is probably contributed by the N-terminal region (438 residues). Our EM class average images of Rpn1 and Rpn2 also show some similarities with the Blm10 protein of S. cerevisiae, an activator that binds to the 20S proteasome, and is composed almost entirely of HEAT repeats 28. In negatively stained class averages, isolated Blm10 molecules exhibit a variety of coiled shapes of a filament of about the same thickness as we observe for Rpn1 and Rpn2 29. However, its shape appears to be specifically defined when bound to the distal surface of the 20S particle 30.

As to the deployment of Rpn1 and Rpn2 in the 19S particle, two models are currently in play. In one of them, based on AFM and cross-linking 16, these proteins’ toroids stack axially through the center of the ATPase ring, with Rpn2 making contact with the apical surface of 20S and Rpn1 protruding on the distal side. Size considerations of the axial holes through AAA+ hexamers of known structure indicate that if the Rpt ring is similarly configured, it cannot accommodate a 6 nm ring; for instance, in FtsH (PDB: 2DHR 31) and P97 (PDB: 1R7R 32), whose AAA+ modules are ~ 45% sequence-similar to Rpt1, this hole is only ~ 2 nm across, widening to ~ 4 nm at the distal surface. However, it is not ruled out that Rpn2 may adjust its shape when incorporated into the 19S particle; for example β-karyopherin, another alpha-solenoid, exhibits considerable plasticity 25.

Alternatively, in their negative stain reconstruction of the human 26S proteasome, da Fonseca & Morris assigned an extended peripheral density in the base region to Rpn1 or Rpn2 19. Similar densities were observed in an earlier cryo-EM analysis of the 26S proteasome 18. This position is also in line with a proposal based on comparing averaged side-views of the 26S proteasome with those of the 20S-PAN ATPase complex, which has no Rpn1 or Rpn2 33. Part of the peripheral density of da Fonseca & Morris (their Figure 4a-ii) appears to be approximately annular and about 6 nm in diameter, raising the possibility that it might represent the PC repeat region of Rpn1 or Rpn2. However, settling this important issue will require more detailed structural information, of which cryo-EM at higher resolution and/or specific labeling experiments, appear to be likely potential sources.

Acknowledgments

We thank Dr A. V. Kajava for providing PDB coordinates for his models. This work was supported by the Intramural Research Program of NIAMS and by grants from the Israel Science Foundation and the USA-Israel Binational Science Foundation to M.G.

Footnotes

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