| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 282, Issue 8, 5545-5550, February 23, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
-Solenoid Model of HET-s Prion Fibrils*
2
From the
Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, and ¶Laboratoire de Génétique Moléculaire des Champignons, Institut de Biochimie et de Génétique Cellulaire, UMR 5095 Centre National de la Recherche Scientifique/Université de Bordeaux 2, 33077 Bordeaux, France
Received for publication, December 14, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
|---|
-solenoids with two coils per subunit, we examined fibrils by electron microscopy. Electron diffraction gave a prominent meridional reflection at (0.47 nm)-1, indicative of cross- structure, as predicted. STEM (scanning transmission electron microscopy) mass-per-unit-length measurements yielded 1.02 ± 0.16 subunits per 0.94 nm, in agreement with the model prediction (1 subunit per 0.94 nm). This is half the packing density of 
1 subunit per 0.47 nm previously obtained for fibrils of the yeast prion proteins, Ure2p and Sup35p, whence it follows that the respective amyloid architectures are basically different. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
|---|
-sheet content (4, 5). This material shows infectivity when introduced into naïve, i.e. uninfected mycelia (5). HET-s has two domains: residues 1 to approximately 220, which form a well folded and presumably globular domain; and the C-terminal prion domain (6, 7). In the soluble state of HET-s, its prion domain is unfolded. This property is shared by the yeast prion proteins Ure2p (8) and Sup35p (9, 10), whose prion domains are larger, enriched in Asn and Gln residues, and located at their N termini. In the absence of the remainder of HET-s, its prion domain has the propensity to fibrillize and to infect (5, 6, 11). These are also properties that HET-s holds in common with Ure2p and Sup35p (12–14). When expressed in P. anserina, the HET-s prion domain alone can propagate the prion, but is not functional in heterokaryon incompatibility. To restore this function, at least parts of its N-terminal domain are needed (15).
Recently, a -solenoid model has been proposed for HET-s-(218–289) fibrils, based on mutational analysis, hydrogen/deuterium exchange, and solid-state NMR spectroscopy (11, 16). Generically, solenoid proteins are axially coiled structures with each coil containing a set of secondary structure elements (17, 18). In -solenoids, these elements are -strands and turns; special cases are the -roll, with two strands and turns per coil, and the -helix with three strands and turns per coil (17, 18, 19). Several -solenoid models have been proposed for different amyloids (20, 21). Such models may be classified in terms of the number of protofibrils per fibril; the number of coils per subunit in a protofibril; and the particular -solenoid fold (22). The model proposed for HET-s fibrils envisages a single protofibril and a -roll fold with two coils per subunit (Fig. 1a; 16). The observation that the HET-s prion domain sequence has two similar subdomains was accommodated by postulating that each subdomain forms a coil of the solenoid, and the experimental determination that each subdomain has two -strands led to the proposed -roll fold (Fig. 1a).
This model makes predictions that are testable, i.e. the presence of cross- structure in the fibril; and that its axial packing density should correspond to one subunit per 0.94 nm. Cross- structure may be detected by electron diffraction (23, 24). Axial packing density may be determined from mass-per-unit-length measurements made by dark-field scanning transmission electron microscopy (STEM)3 (25, 26). In this study, we have applied these techniques to fibrils of the prion domain HET-s-(218–289), and of HET-s-(157–289), a construct containing an additional segment that, when appended to the prion domain, restores competence in heterokaryon incompatibility (15). Our observations lend strong support to the model.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
|---|
Preparation of HET-s-(218–289) and HET-s-(157–289) and Fibril Formation—The histidine-tagged polypeptides were expressed in E. coli BL21(DE3) pLysS and isolated, as has been described for full-length HET-s (4). Both polypeptides were pure as judged by SDS gels stained with Coomassie Blue. Mass spectrometry was used to verify their identities, but showed that the N-terminal methionine was cleaved off from HET-s-(218–289), resulting in molecular masses of 8519.4 Da (HET-s-(218–289)) and 15189.8 Da (HET-s-(157–289)). For fibril assembly, denaturant-containing buffer was exchanged to buffer without denaturants at the desired pH value. For fibril formation at neutral pH, a buffer of 20 mM phosphate, 150 mM NaCl, pH 7.4 was used, whereas fibril formation at lower pH values was performed either in 50 mM phosphate at pH 2.0 or in 20 mM citrate at pH 2.0, pH 3.3, or pH 3.9, respectively. At neutral or near-neutral pH values, fibril formation occurs rapidly (minutes to hours); at lower pH values (pH 2), it is much slower (days to weeks).
Conventional Transmission Electron Microscopy and Electron Diffraction—For negative staining, samples were sonicated in a bath sonicator (Crest Tru-Sweep) for 10–20 min, adsorbed to freshly glow-discharged carbon-coated grids, rinsed with water, and stained with 1% uranyl acetate. Sonication was found to be necessary for reproducible dispersal of singlet fibrils. Micrographs were recorded on a Philips CM120 microscope. For electron diffraction, specimens were changed to water by ultracentrifugation (20,0000 x g, 30 min), immediately embedded in sucrose, and diffraction patterns recorded from areas of 3 µm diameter at low electron doses of 1–4 e/Å2 on a Philips CM120 microscope. Spacing were calibrated with graphitized carbon and evaporated thallous chloride.
Scanning Transmission Electron Microscopy—Freshly prepared fibrils were lightly sonicated (5 s, 60 watts) and checked for optimal length and dispersal by negative staining as described above. Samples deemed to be adequate were frozen rapidly in liquid nitrogen and stored until further processing. Freeze-dried specimens were prepared according to the standard method of the Brookhaven STEM facility (www.biology.bnl.gov/stem/stem.html). Briefly, titanium grids were coated with a thick holey (5–10 µm) carbon film that supported a thin (2–3 nm) continuous carbon film. The thin carbon film was prepared by ultra-high vacuum evaporation onto a freshly cleaved crystal of rock salt and was kept under water and not exposed to air at any point to avoid unspecific adsorption of material. 2 µl of tobacco mosaic virus solution (100 µg/ml) was injected into the drop on the grid and incubated for 1 min. After washing with water four times, 2 µl of the specimen was injected into the drop and incubated for 1 min. Ten more washes in water were performed, and after the final wash, the grid was pinched between the two pieces of filter paper (leaving a retained layer of solution of less than 1 µm) and immediately plunged into liquid nitrogen slush. The grid was transferred to an ion-pumped freeze dryer, freeze-dried overnight by gradual warming to -80 °C and transferred under vacuum to the STEM. Grids were scanned at -150 °C on a custom-built STEM at 40 kV with a probe focused to 0.25 nm with a dwell time of 30 µs/pixel.
Analysis of STEM Images—Digital dark-field micrographs of freeze-dried specimens were recorded with 512 x 512 pixels at raster steps of 1.0 or 2.0 nm per pixel. The images were analyzed using the PCMass (available from the Brookhaven STEM resource; 28) and PIC (29) programs. Boxes of 25 nm length and appropriate width (16 nm for singlet HET-s fibrils and 22 nm for tobacco mosaic virus particles and thicker HET-s fibrils) were used for the measurements. The resulting data were normalized to the known mass-per-unit-length of tobacco mosaic virus (131.4 kDa/nm).
Histograms were calculated with 1-kDa/nm bins. A Gaussian distribution was then fitted to the main peak using the Marquardt-Levenberg algorithm, as implemented in SigmaPlot (Systat Software). If smaller peaks corresponding to doublet and triplet fibrils were present, they were fitted either by including additional independent Gaussian components or by fitting Gaussians to the residual distribution left after subtracting the first fitted Gaussian. The results are given in Table 1. The more complex distribution found for fibrils at pH 2.0 was fitted, based on the assumption that the largest peak represented triplet fibrils and that the other masses observed were different multiples (doublets to septuplets) of the same basic fibril. In this case, all Gaussian components were assigned corresponding width and peaks (mass-per-unitlength values). The number of free parameters was thereby reduced to 8 from the 18 that would be needed if all the Gaussians were to be fitted independently.
|
26 kDa/nm) were selected for analysis. Two examples are shown in supplemental Fig. 2a.
|
1.87 x D(cf. supplemental Fig. 2b). |
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
|---|
2 and 4, which have 11 residues, corresponding to a strand length of 3.85 nm and a short dimension given by stacking two -sheets (2 nm). Allowing some uncertainty in edge detection in the micrographs and the possibility of additional contributions to fibril width from flexible inter-strand loops and ends (31), these numbers are compatible with the measured width of HET-s-(218–289) fibrils. The greater width of HET-s-(157–289) fibrils presumably reflects the presence of the N-terminal appendages. Measurement of diameters from unstained STEM images yielded 4.9 ± 0.4 nm and 5.8 ± 0.4 nm for HET-s-(218–289) and HET-s-(157–289), respectively, in good agreement with the data from negative stain. No systematic variations in width along a given fibril were observed for either construct.
Fibrils of both constructs have a marked tendency to bundle into large cable-like structures with diameters of 20 to >200 nm (supplemental Fig. 1; 6, 15). The average center-to-center spacing of fibrils in a bundle may be measured in terms of spacings of reflections in diffraction patterns calculated from these images. In this way, we measured a spacing of 4.9 ± 0.3 nm for bundles of HET-s-(218–289) fibrils (supplemental Fig. 1a). Again, this number is in good agreement with diameters estimated from negatively stained as well as unstained STEM images of singlet fibrils.
Electron diffraction shows a strong meridional reflection at a spacing of (0.47 nm)-1 with both specimens, establishing the presence of cross- structure (Fig. 1, e and f; supplemental Fig. 1). Images of the specimen areas that yielded these electron diffraction patterns (e.g. Fig. 1f) showed that they contained bundles of HET-s fibrils with diameters of 50–100 nm (supplemental Fig. 1a). Electron diffraction from single bundles confirmed the meridional position of the (0.47 nm)-1 reflection (supplemental Fig. 1, b and c).
In STEM analysis of HET-s-(218–289) fibrils prepared at pH 7.4 (Fig. 1g), the distribution of mass-per-unit-length measurements has a single peak at 9.20 ± 1.44 kDa/nm (S.D.). With a subunit mass of 8.52 kDa, this corresponds to 1.02 ± 0.16 subunits per 0.94 nm (cf. Table 1 for a summary of all STEM measurements). The data for HET-s-(157–289) fibrils prepared at pH 7.4 (Fig. 1h) have a major peak at 13.8 ± 2.02 kDa/nm, corresponding to 0.85 ± 0.13 subunits per 0.94 nm (the subunit mass is 15.19 kDa), with a few higher values corresponding to bundles of two or three fibrils (Table 1).
Our primary interest was in fibrils formed at near-neutral pH, the same condition as was used in the studies that led to the current model. However, in view of the tendency of the fibrils to aggregate (which was eventually resolved, see above), we also explored other ionic conditions, including preparations made at acidic pH (Fig. 2). Fibrils formed at pH 3.9 and 3.3 were found to have less tendency to aggregate, and these preparations were also subjected to STEM mass analysis, yielding major peaks at 9.00 ± 1.53 kDa/nm (0.99 ± 0.17 subunits per 0.94 nm) and 9.52 ± 1.18 kDa/nm (1.05 ± 0.13 subunits per 0.94 nm), respectively, in addition to small amounts of doublet and triplet fibrils (Fig. 2, a and b; Table 1). Thus, to within experimental error, these fibrils have the same axial packing density as fibrils assembled at neutral pH.
We also made some observations on fibrils prepared at pH 2.0. Negative staining showed that they vary in width, but the narrowest have diameters of 8–9 nm and thus are thicker than singlet fibrils formed at higher pHs. Under favorable staining conditions, these minimum-size pH 2 fibrils appear to consist of two or three narrower fibrils with diameters of 4–5 nm (e.g. Fig. 2d; blown-up micrograph at right). These fibrils (the majority; T in Fig. 2c) vary little in width along their lengths. However, other fibrils are wider and exhibit periodic variations in projected width (H in Fig. 2c; arrow in Fig. 2d). STEM mass-per-unit-length data for HET-s-(218–289) fibrils made at pH 2.0 had a major peak at 25.8 ± 3.4 kDa/nm (Fig. 2d). The simplest interpretation of this peak is that it represents triplets of fibrils that, individually, have the same axial density as fibrils prepared at pH 7.4, 3.9, or 3.3. A singlet HET-s-(218–289) fibril at pH 2.0 would have 0.95 ± 0.22 subunits per 0.94 nm. The denser fibrils appear to be mostly bundles of six singlet fibrils (or two triplets; Fig. 2c, histogram). The interpretation that the pH 2 fibrils are mainly triplets and pairs of triplets is consistent with the observation that the putatively triplet fibrils do not vary much in projected width; this is a property of three-stranded ropes, where the individual strands have approximately circular cross-sections (supplemental Fig. 2b). However, the widths of the peaks in the STEM mass data for pH 2 fibrils are greater than those obtained at other pHs, and other interpretations of these data are possible.
The pH at which HET-s prion domain fibrils are grown appears to influence the interaction between fibrils but not to change the overall architecture of singlet fibrils. The observation that fibrils are more dispersed between pH 3.3 and 3.9 than at neutral pH suggests that interactions between charged side chains are involved in their aggregation. There are 8 negatively charged residues and one histidine residue (plus 6 histidines in the his-tag) in the HET-s-(218–289) sequence that could have a pKa value in this range, and all except one are predicted to be on the surface of the -roll or in flexible loops (16, 31).
In summary, mass-per-unit-length data impose a stringent constraint on viable fibril models, when the subunit mass is known. The STEM data obtained for fibrils, assembled from both constructs near neutral pH, and also for HET-s-(218–289) fibrils at acidic pHs, are consistent with the value (1.0 subunits per 0.94 nm) predicted in the stacked -solenoid model of Ritter et al. (16). The fact that appending an additional N-terminal 50 residues to the 72-residue prion domain did not alter the axial packing density in the fibril is consistent with the proposition that HET-s prion domains form an amyloid backbone that is decorated peripherally with other domains, if present. A similar backbone/periphery arrangement has been found in yeast prion filaments of Ure2p and Sup35p (10, 32–34).
|
-solenoid model for HET-s (16), it is also clear that this model does not apply to the yeast prion fibrils.
An alternative model, called the superpleated -structure (35) or alternatively, the stacked -serpentine (Fig. 1b), represents a class of cross- fibril structures that satisfies the massper-unit-length data on Ure2p fibrils and most of the Sup35p fibril subtypes. Corresponding -strands, in successive subunits, stack axially to generate parallel -sheets. Topologically, this model differs from that of Ritter et al. (16) in terms of its serpentine fold and in having one subunit (not half a subunit) per 0.47 nm layer.
There are other reasons to suspect that HET-s fibrils differ structurally from those of the yeast prions. The amino acid compositions of the latter prion domains are rich in uncharged polar residues (Asn, Gln) and have a relative dearth of hydrophobic and charged residues. In these respects, they differ from the HET-s prion domain, which is rich in charged residues, consistent with a higher ratio of surface-exposed residues to buried residues, as in the -solenoid model. In contrast, this ratio should be lower for -serpentines with three or more strands per subunit (35). Moreover, there is no reason to suppose that the prion domains of Ure2p or Sup35p subdivide into two (or more) similar subdomains, which is a key consideration in the proposal of two coils per prion domain in the HET-s model.
Finally, we note that the Ure2p and Sup35p prion domains are redundant, as indicated by their prionogenic properties being unaffected by deletions (36, 37) or sequence randomization (37, 38). In contrast, the only portion of HET-s that tolerates deletions is the central loop (16; Fig. 1a). Furthermore, the yeast prions exhibit strain variants (12–14, 39, 40) that have been correlated with amyloid polymorphisms (12, 22, 27, 34, 41). The -serpentine model readily explains such polymorphisms; for instance, with the serpentine deriving from different segments of the prion domain. It remains to be seen whether HET-s fibrils also exhibit similar polymorphisms and strain variations. In this respect, the two-coil -solenoid appears to be less amenable to polymorphism.
|
FOOTNOTES |
|---|
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.
1 Supported by the groupement d'intérêt scientifique prion (Ministère de la Recherche, France).
2 To whom correspondence should be addressed: Laboratory of Structural Biology, National Institutes of Health, Bldg. 50, Rm. 1517, 50 South Dr. MSC 8025, Bethesda, MD 20892-8025. Tel.: 301-496-0132; Fax: 301-443-7651; E-mail: Alasdair_Steven{at}nih.gov.
3 The abbreviation used is: STEM, scanning transmission electron microscopy.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Wasmer, A. Lange, H. Van Melckebeke, A. B. Siemer, R. Riek, and B. H. Meier Amyloid Fibrils of the HET-s(218-289) Prion Form a {beta} Solenoid with a Triangular Hydrophobic Core Science, March 14, 2008; 319(5869): 1523 - 1526. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Malato, S. Dos Reis, L. Benkemoun, R. Sabate, and S. J. Saupe Role of Hsp104 in the Propagation and Inheritance of the [Het-s] Prion Mol. Biol. Cell, December 1, 2007; 18(12): 4803 - 4812. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
