JMB
Available online at www.sciencedirect.com
@
SCIENCE DIRECT®
AP
Reversible Amyloid Formation by the p53 Tetramerization Domain and a Cancer- associated Mutant
Amanda S. Lee1;, Charles Galea1;, Enrico L. DiGiammarino1 Bokkyoo Jun2, Gopal Murti3, Raul C. Ribeiro4, Gerard Zambetti5,6 Christian P. Schultz2 and Richard W. Kriwacki1,6*
1Department of Structural Biology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis TN 38105, USA
2Bruker Optics, Inc., 19 Fortune Drive, Billerica, MA 01821,USA
3Division of Virology/Department of Infectious Diseases, St. Jude Children’s Research Hospital 332 North Lauderdale Street Memphis, TN 38105, USA
4International Outreach Program, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis TN 38105, USA
5Department of Biochemistry St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105 USA
6Department of Molecular Sciences, University of Tennessee Health Sciences Center, 790 Madison Avenue Memphis, TN 38163, USA
The tetramerization domain for wild-type p53 (p53tet-wt) and a p53 mutant, R337H (p53tet-R337H), associated with adrenocortical carcinoma (ACC) in children, can be converted from the soluble native state to amy- loid-like fibrils under certain conditions. Circular dichroism, Fourier transform infrared spectroscopy and staining with Congo red and thiofla- vin T showed that p53tet-wt and p53tet-R337H adopt an alternative ß- sheet conformation (p53tet-wt-ß and p53tet-R337H-B, respectively), characteristic of amyloid-like fibrils, when incubated at pH 4.0 and elev- ated temperatures. Electron micrographs showed that the alternative con- formations for p53tet-wt (p53tet-wt-B) and p53tet-R337H (p53tet-R337H- B) were supramolecular structures best described as “molecular ribbons”. FT-IR analysis demonstrated that the mechanism of amyloid-like fibril for- mation involved unfolding of the p53tet-wt ß-strands, followed by unfolding of the a-helices, followed finally by formation of ß-strand-con- taining structures that other methods showed were amyloid-like ribbons. The mutant, p53tet-R337H, had a significantly higher propensity to form amyloid-like fibrils. Both p53tet-wt (pH 4.0) and p53tet-R337H (pH 4.0 and 5.0), when incubated at room temperature (22 ℃) for one month, were converted to molecular ribbons. In addition, p53tet-R337H, and not p53tet-wt, readily formed ribbons at pH 4.0 and 37 ℃ over 20 hours. Inter- estingly, unlike other amyloid-forming proteins, p53tet-wt-ß and p53tet- R337H-ß disassembled and refolded to the native tetramer conformation when the solution pH was raised from 4.0 to 8.5. Although fibril formation at pH 4.0 was concentration and temperature-dependent, fibril disassem- bly at pH 8.5 was independent of both. Finally, we propose that the sig- nificantly higher propensity of the mutant to form ribbons, compared to the wild-type, may provide a possible mechanism for the observed nuclear accumulation of p53 in ACC cells and other cancerous cells.
@ 2003 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: p53; tetramerization domain; amyloid-like fibril; amyloidogenesis; protein aggregation
* These authors contributed equally to this work. Present address: E. L. DiGiammarino, Department of Chemistry, The University of Alabama in Huntsville, 229 Materials Science Building, Huntsville, AL 35899, USA.
Abbreviations used: ACC, adrenocortical carcinoma; CD, circular dichroism; CR, Congo red; 2D TROSY, two- dimensional transverse relaxation optimized spectroscopy; EM, electron microscopy; FT-IR, Fourier transform infrared spectroscopy; Gdn-HCl, guanidine hydrochloride; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight-mass spectrometry; p53, full-length p53 protein; p53tet, p53 tetramerization domain; p53tet-wt, wild-type p53 tetramerization domain; p53tet-wt-ß, ß-structure form of p53tet-wt; p53tet-R337H, mutant (R337H) form of p53 tetramerization domain; p53tet-R337H-B, ß-structure form of p53tet-R337H; ThT, thioflavin T.
E-mail address of the corresponding author: richard.kriwacki@stjude.org
Introduction
A number of human diseases including Alzhei- mer’s disease, diabetes type II, and spongiform encephalopathies are characterized by deposition in tissues of protein aggregates known as amyloid fibrils.1-4 Also, proteins not associated with human disease have been shown to convert in vitro to higher order structures with the same characteristics as disease-associated amyloid fibrils.5-7 These observations support the view that formation of amyloid fibrils is a generic property of peptides and proteins.5,7,8 Mutant forms of the tumor suppressor protein p53 are known to accumulate in many types of cancer cells.9,10 One example of this is a p53 mutant (R337H) associated with adrenocortical carcinoma (ACC) in children.11 Past studies of the tetramerization domain of a p53 mutant, R337H (p53tet-R337H), have shown that substitution of R337 by histidine results in a pH-dependent instability of this domain.12 In the native protein residue R337 forms a stable salt bridge with residue D352 of an adjacent p53 sub- unit. It has been shown that substitution of this residue by histidine weakens this salt bridge in a pH-dependent manner. We report here that under certain conditions the tetramerization domain of wild-type p53 (p53tet-wt) and the mutant (p53tet- R337H) can form amyloid-like structures, which we have characterized as “molecular ribbons”. Further, we define the structural steps leading to the formation of p53tet ribbons and show that the process can be reversed through a change in pH. We propose that the significantly higher propensity of the mutant to form ribbons, compared to the
wild-type, may provide a possible mechanism for the observed nuclear accumulation of p53 in ACC cells and other cancerous cells.
Results
Formation of p53tet amyloid-like fibrils
Initially, we identified conditions that convert native p53tet-wt and p53tet-R337H into amyloid- like fibrils. Thermal denaturation of p53tet-wt and p53tet-R337H at pH 4.0 and at high or low salt con- centrations was accompanied by changes in circu- lar dichroism (CD) spectra corresponding to an increase in the magnitude of negative ellipticity at 222 nm at temperatures above the melting tem- peratures (Figure 1(a)-(d)). The CD spectrum of the native conformation of p53tet-wt at 25 ℃ (Figure 2(a)) was dominated by «-helical features (minima at 222 nm and 208 nm). At 75 ℃ p53tet- wt was denatured (Figure 2(b)), as shown by the absence of a-helical and ß-strand characteristics in the CD spectrum. The spectrum obtained at 95 ℃ provided evidence for an alternative conformation (Figure 2(c); termed p53tet-wt-ß) comprised largely of ß-strands (minimum near 216 nm) that formed after denaturation of the native structure. p53tet- R337H behaved similarly as a function of tempera- ture (Figure 1(c) and (d)). However, formation of the alternative conformation (p53tet-R337H-) initially occurred at a lower temperature for p53tet-R337H (above 61 ℃) compared to p53tet-wt (above 72 ℃), indicating significant differences in the thermostabilities of the two domains.13 The
Fraction Unfolded Fraction Unfolded
1.0
(a)
1.0
(b)
0.5
0.5
0.0
0.0
1.0
(c)
1.0
(d)
0.5
0.5
0.0
0.0
20
30 40 50 60 70 80
90
20
30
40
50
60
70
80
90
Temperature (C)
Temperature (C)
[0] x 10-3 (deg cm2/dmol)
0
(a)
0
(b)
-1
-1
-2
222 nm
incr.
-2
-3
-4
208 nm
temp. - 3
-4
-5
-5
-6
25 ℃
-6
75 ℃
-7
200 210 220 230 240 250 260 Wavelength (nm)
-7
200
210
220
30 240 25
260
incr. Wavelength (nm)
temp.
[O] x 10-3 (deg cm2/dmol)
0
(c)
0
(d)
-1
-1
-2
216 nm
decr …- 2
216 nm
-3
temp.
-3
-4
-5
-5
-6
95 ℃
-6
25 ℃
-7
-7
200 210 220 230 240 250 260 Wavelength (nm)
200 210 220 230 240 250 260 Wavelength (nm)
unfolding temperatures for native p53tet-wt and p53tet-R337H at pH 4.0 were 69℃ and 46 ℃, respectively. Further, p53tet-wt-ß and p53tet- R337H-ß were stable at temperatures below 95 ℃ (Figure 2(d) and data not shown). Interestingly, p53tet-wt and p53tet-R337H did not readily form this conformer at high temperatures at pH 5.0, or higher. These results show that p53tet-wt and p53tet-R337H undergo a structural change from their native structure (Figure 3) to an alternative ß-sheet conformation, characteristic of amyloid- fibrils, when incubated at pH 4.0 and elevated tem- peratures. Carboxylate groups undergo protona- tion in this pH range. It is possible that protonation of Asp and/or Glu residues at pH 4.0 destabilizes the native structure and promotes the formation of non-native, ß-aggregates.
FT-IR analysis of p53tet amyloid-like fibrils
Since CD spectroscopy is not very sensitive in detecting small populations of ß-sheet confor- mations, Fourier transform infrared (FT-IR) spec- troscopy was used to gain further insight into the thermal denaturation13 and structural transition14 of p53tet-wt and p53tet-R337H to the alternative B-strand-rich conformation at pH 4.0. Results show that the change in the relative integrated intensity of the amide I band between 1678 cm-1 and 1631 cm-1 for p53tet-wt, which contained absorbances for native peptide conformations,15 exhibited a temperature dependence similar to that of the CD signal at 222 nm (Figure 4). The FT- IR spectrum acquired for p53tet-wt at 25 ℃ (Figure 5(a)) was dominated by a strong absorbance at
R 337
D 352
0.8
0.7
Intensity
incr.
0.6
a
temp.
b
0.5
d
decr.
C
0.4
temp.
0.3
0
20
40
60
80
100
Temperature ( ℃)
(a)
1654 cm-1
(b)
0.030
0.030
1645 cm-1
Absorbance
incr.
0.020
temp.
0.020
1616 cm-1
0.010
0.010
0.000
25 ℃
0.000
65 ℃
1700
1660
1620
1580
1700
1660
1620
1580
Wavelength (cm-1)
incr.
Wavelength (cm-1)
(c)
1616 cm-1
temp.
1613 cm-1
(d)
0.030
Absorbance
decr.
0.030
0.020
temp.
1683 cm-1
0.020
1683 cm-1
0.010
0.010
0.000
95 ℃
0.000
25 ℃
1700
1660
1620
1580
1700
1660
1620
1580
Wavelength (cm-1)
Wavelength (cm-1)
1654 cm-1 corresponding to «-helices. Upon heat- ing from 25℃ to 65℃ this band shifted to 1645 cm-1, corresponding to random peptide con- formations, consistent with the denaturation of the & helices in p53tet-wt (Figure 5(b)). As temperature was increased to 95℃, the intensity of bands between 1678 cm-1 and 1631 cm-1 decreased further; new bands appearing at 1683 cm-1 and 1616 cm-1 (Figure 5(b) were indicative of ß- strand-containing structures with strong intermole- cular hydrogen bonds.7 In agreement with the CD analysis, FT-IR spectra of p53tet-wt show that the ß structures that form at 95 ℃ were stable when the temperature was reduced to 25 ℃ (Figure 5(c) and (d)). Similar results were obtained for p53tet- R337H (data not shown) except that denaturation and formation of the alternative conformation occur at lower temperatures. Therefore, FT-IR spec- tra confirm the structural transition of p53tet-wt and p53tet-R337H from the native conformer to a denatured state, followed by a stable ß-strand-rich conformation.
EM analysis of morphology of p53tet amyloid- like fibrils
Electron micrographs showed that the alterna- tive conformations for p53tet-wt (p53tet-wt-ß) and p53tet-R337H (p53tet-R337H-ß) were supramole- cular structures best described as molecular rib- bons (Figure 6(a) and (b)), which were several microns in length, approximately 10 nm wide, and
approximately 1-2 nm thick. The morphology of these ribbons differed from classical amyloid fibrils, which have a cylindrical cross-section, and may be structural precursors to amyloid fibrils.16 Importantly, electron microscopy (EM) showed that both p53tet-wt and p53tet-R337H, when incu- bated at room temperature (22 ℃) for one month, were converted to molecular ribbons (data not shown). Ribbons were observed for p53tet-wt incu- bated at pH 4.0, and for p53tet-R337H incubated at pH 4.0 and 5.0. In addition, further studies have shown that p53tet-R337H, and not p53tet-wt, readily forms ribbons at pH 4.0 and 37 ℃ over 20 hours (data not shown). These observations demonstrate that extreme temperatures are not required for the formation of p53tet ribbons. The similarity of the CD and FT-IR spectral features for p53tet-wt-ß and p53tet-R337H-ß to those of amyloid fibrils formed from a variety of proteins7,17-19 suggested that they have a similar stacking of ß-strands but may differ in how these are organized on a larger structural scale.
Reaction of p53tet fibrils with Congo red and thioflavin T
To further characterize p53tet-wt-ß and p53tet- R337H-ß fibrils, we performed solution assays using the chemical dyes, Congo red20 (CR) and thioflavin T (ThT).21 Samples of p53tet-wt and p53tet-R337H (pH 4.0) were either maintained at 25 ℃ or were incubated at an elevated temperature
(a)
0.2 um
(b)
0.2 um
where the temperature was incrementally increased from 25 ℃ to 95℃ over 60 minutes. These protein solutions were then treated with CR or ThT. Both samples treated at high temperature and stained with CR exhibited a red-shift of absor- bance wavelength from 495 nm to 503 nm, which is characteristic of amyloid fibrils20 (data not shown). The absorbance wavelength did not shift when CR was added to the p53tet samples incu- bated at 25 ℃. Further, both p53tet samples treated at 95 ℃ caused the fluorescence emission intensity of ThT to increase while samples treated at 25 ℃ did not exhibit a change in fluorescence. Therefore, p53tet samples treated at 95 ℃ exhibited spectral changes characteristic of amyloid fibrils when trea- ted with Congo red or ThT and confirmed our
results from CD and FT-IR spectroscopy and EM. Together, these results demonstrated that p53tet- wt and p53tet-R337H form amyloid-like molecular ribbons when treated at low pH. High tempera- tures promote rapid formation of ribbons; how- ever, ribbons are observed to form slowly at room temperature.
Stability of p53tet-wt and p53tet-R337H amyloid-like fibrils
Results from experiments involving denatura- tion with guanidine hydrochloride (Gdn-HCI) demonstrated that p53tet-R337H was considerably less stable than p53tet-wt at low pH (Figure 7(a) and (b)), confirming results based on thermal
Fraction Unfolded
1.0
(a)
1.0
(b)
0.5
0.5
0.0
0.0
0
1
2
3
4
5
6
0
1
2
3
4
5
6
[0] x 10-3 (deg cm2/dmol)
-2
(c)
-2
(d)
-3
-3
-4
-4
-5
-5
-6
-6
-7
-7
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
[Gdn·HCI] (M)
[Gdn·HCI] (M)
[0] x 10-3 (deg cm2/dmol)
10
(a)
0
-10
-20
-30
-40
200
210
220
230
240
250
260
Wavelength (nm)
(b)
115.0
15N (ppm)
Native
125.0
pH 6.0
25 ℃
9.0
8.0
1H (ppm)
7.0
(c)
115.0
15N (ppm)
fibers
125.0
pH 4.0
35 ℃
9.0
8.0
1H (ppm)
7.0
(d)
115.0
15N (ppm)
Native
125.0
pH 6.0
25 ℃
9.0
8.0
7.0
1H (ppm)
denaturation (Figure 1(a) and (b)). The midpoint of the chemical denaturation curve for p53tet-wt was observed at 2.0 M Gdn-HCI (Figure 7(a)) and that for p53tet-R337H was observed at 0.9 M Gdn-HCI (Figure 7(b)). These curves exhibited the character- istic sigmoid shape associated with cooperative unfolding of proteins.22 In contrast, treatment of p53tet-wt-ß and p53tet-R337H-ß with Gdn-HCl revealed denaturation curves (Figure 7(c) and (d)) that were linearly dependent on Gdn-HCl concen- tration. Interestingly, the slopes of lines drawn through these curves were very similar for p53tet- wt-ß and p53tet-R337H-ß, suggesting that similar intermolecular forces stabilized the respective structures. The lack of cooperativity for Gdn-HCI- dependent denaturation of the p53tet-ß structures was consistent with results for other aggregated structures comprised of ß-strands.23
pH-dependent reversibility of fibril formation
In contrast to amyloid fibrils from many sources, which often form irreversibly and are extremely stable,4,24 p53tet-wt-B and p53tet-R337H-B uniquely disassembled and refolded to the native tetramer conformation when the solution pH was raised from 4.0 to 8.5 (for p53tet-R337H, Figure 8(a); for p53tet-wt, data not shown). Progressive structural changes were observed between pH 4.0 and 8.5 but complete disassembly and refolding of the native conformation occurred only when the pH reached 8.5 (Figure 8(a)). Interestingly, the dis- assembled and refolded species retained no “his- tory” of its aggregated past, as evidenced by retention of the native CD spectrum after recycling pH from 8.5 to 4.0 (Figure 8(a), green versus magenta traces).
Concentration dependence of fibril formation
We investigated the concentration dependence of assembly and disassembly by performing exper- iments at high and low protein concentrations (3.6 mM and 0.4 mM, pH 4.0) using NMR spec- troscopy to monitor protein structure. The 2D transverse relaxation optimized spectroscopy (TROSY)25 spectrum of “native” 15N-labeled p53tet-R337H at a concentration of 3.6 mM is shown in Figure 8(b). This spectrum revealed the appropriate number of well-resolved, sharp reson- ances corresponding to the tetrameric, native-like structure.12 Surprisingly, at 35 ℃, we observed a dramatic reduction in the number of observable resonances in the 2D TROSY spectrum (Figure 8(c)). This spectral change, which occurred well
the pH of this solution was reduced to 4.0 and the temperature increased to 35 ℃ (c), and after the solution pH was raised from 4.0 to 8.5 for 30 minutes and the temperature reduced to 25 ℃. Finally, the solution pH was reduced to 6.0 (d) for comparison with (b).
0.025
(a)
fiber formation
native
protein
Absorbance
0.020
0.015
0.010
0.005
0.000
1700
1680
1660
1640
1620
1600
1580
Wavenumber (cm-1)
Absorbance (x10-3) cm2
0.2
(b)
fiber formation
0.1
0.0
-0.1
1684
1639
-0.2
native ß-sheet
-0.3
1645
1654
-0.4
a-helices
1616
1700
1680
1660
1640
1620
1600
1580
Wavenumber (cm-1)
below the thermal melting temperature of 46 ℃ and well below the fibril transition temperature of 62 ℃ observed using NMR at a lower protein con- centration (0.4 mM, data not shown), was indica- tive of fibril formation, as was confirmed by staining with CR and ThT using an unlabeled pro- tein sample treated similarly (data not shown). Approximately 25 backbone amide resonances appear in Figure 8(c) out of a total of 54 possible resonances for the p53tet-R337H domain under study. The amide moieties that give rise to these resonances must exhibit local flexibility and, there- fore, are probably not integral to the rigid ß-strand structure of p53tet-R337H ribbons. In contrast to the results for p53tet-R337H, a 3.6 mM solution of 15N-labeled p53tet-wt remained folded as a native tetramer at temperatures up to 60 ℃ (data not shown). The differing propensity of p53tet-wt and p53tet-R337H to form fibrils was likely due to their different thermostabilities. Dobson and co- workers7 have proposed that partially denaturing conditions are required for the formation of amy- loid-like fibrils. Since amyloids are thought to form through self-assembly involving nucleation by precursor “proto-filament” structures,26 the
lower thermostability of p53tet-R337H at 35 ℃ would result in a larger fraction of proto-filament molecules compared with p53tet-wt (Figure 1(a) and (c)). At high protein concentrations these proto-filaments would readily self-associate to form amyloid-like fibrils. On the other hand, at low p53tet-R337H concentrations fibril formation was only observed at significantly higher tempera- tures (Figure 1(c) and (d) and NMR data not shown). Analysis of fibril disassembly by 2D TROSY NMR spectroscopy showed that, unlike fibril formation, fibril disassembly and refolding to the native tetrameric structure at pH 8.5 pro- ceeded independent of concentration and tempera- ture (Figure 8(d)). Gel filtration chromatography confirmed that the product of disassembly of low and high concentrations of p53tet-R337H-ß fibrils exhibited hydrodynamic properties identical to those of the native p53 tetramerization domain (data not shown). Overall, these results showed that while fibril formation at pH 4.0 is dependent upon concentration and temperature, fibril dis- assembly at pH 8.5 is independent of both.
Characterization of fibril formation by FT-IR
As mentioned earlier, the mechanism of amyloid fibril formation, in general, is thought to involve partial unfolding of the native state followed by assembly into ß-strand-containing intermediate proto-filaments that nucleate fibril growth. To gain insight into structural changes that accompanied conversion of native p53tet-wt into amyloid fibrils at pH 4.0, we recorded FT-IR spectra at tempera- tures between 10 ℃ and 95 ℃ in 5 deg. C intervals (Figure 9(a) and (b)). The native structure was maintained between 10 ℃ and 20 ℃ (Figure 9(a) and (b), blue traces). The first structural changes occurred between 25 ℃ and 45 ℃ (Figure 9(a) and (b), green traces), as revealed by spectral changes near 1639 cm-1 easily seen in the second derivative spectra (Figure 9(b)). The decrease in intensity at this wavelength corresponded to loss of native ß- strand secondary structure that stabilizes dimer subunits within the tetramer (Figure 3). This change was followed by the shift of an absorbance band centered at 1654 cm-1 to 1645 cm-1 between 50 ℃ and 70 ℃, which corresponded to unfolding of a-helical secondary structure. Between 65 ℃ and 70 ℃, a new spectral feature appeared at 1616 cm-1 that was characteristic of ß-strands in amyloid fibrils. As the temperature was increased further, this absorbance band and the weaker accompanying band at 1684 cm-1, also character- istic of ß-strands in amyloid fibrils, increased in intensity and eventually dominated the FT-IR spec- trum at 95 ℃ (Figure 9(a)). From these spectra it was possible to deduce the sequence of thermally induced unfolding events which basically involved unfolding of the p53tet-wt ß-strands, followed by the unfolding of the a-helices, followed finally by formation of ß-strand-containing structures that
(a)
35℃
-₿
(b)
+₿
<a +B
-a +BA
50°℃
65℃
(c)
(d)
+BA <a
l
+BA
IF
(e)
pH4++pH8.5
R
1
Gdn.HCI + Dialysis
+BA -a
+BA
Thermal dependence of fibril formation
To understand the thermal constraints on ribbon formation, thermal denaturation was monitored by heating native p53tet-wt (pH 4.0) from 10 ℃ to 35 ℃, 50 ℃ or 65 ℃ followed by cooling to 10 ℃. FT-IR spectra were recorded at 5 deg. C intervals during the heating and cooling steps. These exper- iments (data not shown) revealed structural details of the thermal denaturation and fibril formation processes, as illustrated schematically in Figure 10. Unfolding of the native ß-sheet structure of p53tet-wt was reversible between 10 ℃ and 35 ℃ (Figure 10(a) and (b)). The helical core remained folded during this thermal cycle and amyloid-like ribbons were not observed. Spectral features con- sistent with the formation of a small population of ribbons were observed upon heating to 50 ℃, and to 65 ℃. The FT-IR spectrum for the sample heated to 50 ℃ indicates the presence of some residual helical secondary structure (Figure 10(c)). How- ever, this helical structure gradually disappeared as spectral features associated with p53tet ribbons increased in intensity (Figure 10(e)). In contrast,
the FT-IR spectra for the protein heated to 65 ℃ lacked helical characteristics, indicating that the helical core had unfolded at this temperature. Further, incubation of the sample at 65 ℃ resulted in the conversion of this unfolded protein to rib- bons. (Figure 10(d)). Since FT-IR detected ribbon formation in the presence of some helical structure at 50 ℃ it is possible that the ß-strands, that com- prised ribbons, were initially formed from the ther- mally denatured ß-strands of the tetramer. These studies provide some insight into the mechanism of fibril formation by the p53tet domain. By com- paring spectral changes that occurred during ther- mocycles to different temperatures, it was possible to deduce that discreet, thermally induced struc- tural changes occurred at different temperatures during the unfolding of the p53tet domain and conversion to fibrils (illustrated in Figure 10).
Discussion
Together, these results clearly demonstrate a novel structural form, molecular ribbons, for the tetramerization domain of the p53 tumor suppres- sor protein. Further, a mutant form of this domain that is associated with ACC in children (p53tet-
R337H) exhibited an increased propensity to form molecular ribbons at both high and low tempera- tures. Results from CD, FT-IR, EM and reaction with the diagnostic dyes, Congo red and thioflavin T, demonstrated that the alternative structures for both wild-type and mutant p53tet were very simi- lar to amyloid fibrils formed by other proteins, some of which are associated with human disease.24,27 The observation that about half of the amide moieties in p53tet-R337H ribbons were flex- ible (based on NMR data) implied that the remain- ing 25-30 residues comprised the rigid ß-strands of the amyloid-like molecular ribbons. Further, the similarity of the stability of p53tet-wt-ß and p53tet-R337H-ß to chemical denaturation suggested that the mutant site, residue 337, was not integral to the ribbon structure. Fibril for- mation clearly involved unfolding of p53tet native structure, and appeared to involve two steps: (1) unfolding of the ß-strands on the exterior of the p53tet domain structure28-30 followed by (2) unfolding of the a-helical core of the domain. The native structure of p53tet-R337H is very simi- lar to that of p53tet-wt;12 however, the mutant domain exhibited a lower thermal denaturation temperature than p53tet-wt under fibril-forming conditions (pH 4.0). The observation that p53tet- R337H exhibited a higher propensity to form amy- loid fibrils supports the view that the mechanism of assembly required the generation of non-native, extended polypeptide strands which then assembled into ribbons. We observed rapid ribbon formation for p53tet-R337H at 35 ℃ and slow rib- bon formation for both p53tet-wt and p53tet- R337H at room temperature. These temperatures were well below the midpoints of the thermal melt- ing transitions for both p53tet domains and these results suggest that only a small population of unfolded molecules was required to nucleate con- version of all molecules to the amyloid-like confor- mation. Therefore, high temperatures were not required to form p53tet ribbons when a sufficient number of unfolded protein molecules were pre- sent in solution, or when the process was allowed to proceed for a period of weeks. The observation that p53tet ribbons disassembled and refolded to the native structure at pH 8.5 seems to be a unique feature of these fibrils. Understanding the molecu- lar mechanism of disassembly may, in the future, provide insight into alternative therapeutic strat- egies for treating amyloid diseases.
Finally, it is intriguing to consider the possibility that these results, for an isolated domain under in vitro conditions, may be relevant to the biology of p53, especially cancer-associated mutant forms of this vital tumor suppressor. Mutant forms of p53 are known to accumulate at high levels in cancer cells.9,10 Whether these accumulated forms of p53 adopt amyloid-like structures is unknown. The requirement for low pH to form p53tet fibrils demonstrated in this study would argue that our observations are not relevant to cancer biology. However, some cellular compartments (e.g. endo-
somes associated with protein translocation and lysosomes associated with protein degradation) are characterized by low pH. In addition, previous studies have also shown that low pH is required for the formation of fibrils from the amyloidogenic form of some proteins in vitro.31-36 It has been argued that the expression levels and/or mutation of other genes,37-39 as well as cellular or environ- mental factors,40-44 may enhance the rate of fibril formation in vivo. Further, it is likely that there are other p53 mutants that are unstable under physio- logical conditions, presenting the possibility that some mutant forms of p53 could accumulate in cells as amyloid-like fibrils, potentially leading to cancer or other diseases.
Materials and Methods
Expression and purification of p53tet
Unlabeled and 15N-labeled p53tet-wt and p53tet- R337H were expressed and purified as reported.12 Pro- tein identity was confirmed using MALDI-TOF mass spectrometry.
Formation of p53tet-wt-ß and p53tet-R337H-B
Samples of p53tet-wt-ß and p53tet-R337H-B (0.25- 3.2 mg/ml) dissolved in 20 mM sodium phosphate (pH 4.0), 50 mM NaCl were prepared by gradual heating from 25 ℃ to 95℃ in a thermostated water bath by increasing the temperature in 10 deg. C increments over one hour followed by cooling to 25 ℃. Fibril samples were stored at 4 ℃ or - 20 ℃.
Circular dichroism
CD spectra were recorded using an AVIV model 62A DS circular dichroism spectropolarimeter. Thermal dena- turation curves were obtained by monitoring ellipticity at 222 nm as a function of temperature and converting these values into mole fraction of protein unfolded assuming a two-state unfolding model. CD spectra were obtained using a 0.1 cm path length quartz cell. The reported spectra are an average of three scans and were recorded at various temperatures (15-95 ℃) and pH values (4.0-8.5). Gdn-HCI denaturation curves for native p53tet-wt and p53tet-R337H were recorded using a 1 cm × 1 cm quartz cuvette, and data were collected in 0.12 M increments. Guanidine denaturation curves for p53tet-wt-ß and p53tet-R337H-ß were recorded using a 0.1 cm cuvette, and data were collected in 0.5 M increments.
Infrared spectroscopy
Protein samples (10 mg/ml) for IR experiments were dialyzed four times against a tenfold excess of 20 mM sodium phosphate (pH 7.0), 250 mM NaCl prepared using 2H2O (99%) and anhydrous reagents. pH was adjusted to 4.0 with dilute 2HCl. The final concentration of p53tet-wt and p53tet-R337H was 2 mg/ml. Spectra of p53tet-wt were recorded at 10-95 ℃ in 5 deg. C incre- ments on a Vector 22 FT-IR spectrometer (Bruker Optics, Inc., MA) equipped with a liquid N2-cooled photovoltaic
Mercury-Cadmium-Telluride detector. A sample volume of 5 ul per thermal cycle was analyzed using either a Bio- TransCell™ (between two CaF2 windows with 50 pm path length described previously45) or a BioATRcell™ II (onto the surface of a silicon microwafer).
Electron microscopy
Protein samples (native p53tet-wt and p53tet-R337H, and fibril-containing preparations) were adsorbed to freshly glow-discharged, carbon-coated, electron micro- scope grids and negatively stained with 2% (w/v) phos- photungstic acid (pH 6.4) or 1% (w/v) aqueous uranyl acetate (pH 2.4). The grids were examined in a Philips EM 301 electron microscope operated at 80 kV. Both staining procedures gave similar results and the electron micrographs in Figure 6 were made using the phospho- tungstic acid procedure.
Nuclear magnetic resonance spectroscopy
Two-dimensional 1H-15N TROSY spectra of [15N]p53tet-wt and p53tet-R337H dissolved in 20 mM sodium phosphate (pH 6.0), containing 50 mM NaCl were recorded as described.12 Spectra of [15N]p53tet-wt were recorded at 25-60 ℃ in 5 deg. C increments; spec- tra of [15N]p53tet-R337H were recorded at 25-55 ℃ (0.4 mM) or 25-35 ℃ (3.6 mM) in 5 deg. C increments.
Acknowledgements
Weixing Zhang and Charles Ross are thanked for assistance with NMR experiments and computer support, respectively, and Clive Slaughter and Bill Lewis for mass spectrometry support. Authors declare they have no competing financial interests. This work was supported by the American Leba- nese Syrian Associated Charities, St. Jude Chil- dren’s Research Hospital (SJCRH) Pediatric Oncology Education Program (A.S.L.), American Cancer Society (R.W.K.), National Cancer Institute (R.W.K. and G.Z.), and a Cancer Center (CORE) Support Grant CA 21765 (SJCRH).
References
1. Tan, S. Y. & Pepys, M. B. (1994). Amyloidosis. Histo- pathology, 25, 403-414.
2. Ghetti, B., Piccardo, P., Frangione, B., Bugiani, O., Giaccone, G., Young, K. et al. (1996). Prion protein amyloidosis. Brain Pathol. 6, 127-145.
3. Kelly, J. W. (1997). Amyloid fibril formation and pro- tein misassembly: a structural quest for insights into amyloid and prion diseases. Structure, 5, 595-600.
4. Cohen, F. E. & Prusiner, S. B. (1998). Pathologic con- formations of prion proteins. Annu. Rev. Biochem. 67, 793-819.
5. Guijarro, J. I., Sunde, M., Jones, J. A., Campbell, I. D. & Dobson, C. M. (1998). Amyloid fibril formation by an SH3 domain. Proc. Natl Acad. Sci. USA, 95, 4224-4228.
6. Litvinovich, S. V., Brew, S. A., Aota, S., Akiyama, S. K., Haudenschild, C. & Ingham, K. C. (1998). For-
mation of amyloid-like fibrils by self-association of a partially unfolded fibronectin type III module. J. Mol. Biol. 280, 245-258.
7. Chiti, F., Webster, P., Taddei, N., Clark, A., Stefani, M., Ramponi, G. & Dobson, C. M. (1999). Designing conditions for in vitro formation of amyloid protofila- ments and fibrils. Proc. Natl Acad. Sci. USA, 96, 3590-3594.
8. Dobson, C. M. & Karplus, M. (1999). The fundamen- tals of protein folding: bringing together theory and experiment. Curr. Opin. Struct. Biol. 9, 92-101.
9. Porter, P. L., Gown, A. M., Kramp, S. G. & Coltrera, M. D. (1992). Widespread p53 overexpression in human malignant tumors. An immunohistochemical study using methacarn-fixed, embedded tissue. Am. J. Pathol. 140, 145-153.
10. Bartek, J., Bartkova, J., Vojtesek, B., Staskova, Z., Lukas, J., Rejthar, A. et al. (1991). Aberrant expression of the p53 oncoprotein is a common feature of a wide spectrum of human malignancies. Oncogene, 6, 1699-1703.
11. Ribeiro, R. C., Sandrini, F., Figueiredo, B., Zambetti, G. P., Michalkiewicz, E., Lafferty, A. R. et al. (2001). An inherited p53 mutation that contributes in a tis- sue-specific manner to pediatric adrenal cortical car- cinoma. Proc. Natl Acad. Sci. USA, 98, 9330-9335.
12. DiGiammarino, E. L., Lee, A. S., Cadwell, C., Zhang, W., Bothner, B., Ribeiro, R. C. et al. (2002). A novel mechanism of tumorigenesis involving pH-depen- dent destabilization of a mutant p53 tetramer. Nature Struct. Biol. 9, 12-16.
13. Fabian, H., Schultz, C., Naumann, D., Landt, O., Hahn, U. & Saenger, W. (1993). Secondary structure and temperature-induced unfolding and refolding of ribonuclease T1 in aqueous solution. A Fourier transform infrared spectroscopic study. J. Mol. Biol. 232, 967-981.
14. Schultz, C. P. (2000). Illuminating folding intermedi- ates. Nature Struct. Biol. 7, 7-10.
15. Fabian, H. & Schultz, C. P. (2000). Fourier transform infrared spectroscopy in peptide and protein anal- ysis. In Encylopedia of Analytical Chemistry (Meyers, R. A., ed.), pp. 1-25, Wiley, Chichester.
16. Aggeli, A., Nyrkova, I. A., Bell, M., Harding, R., Car- rick, L., McLeish, T. C. et al. (2001). Hierarchical self- assembly of chiral rod-like molecules as a model for peptide beta sheet tapes, ribbons, fibrils, and fibers. Proc. Natl Acad. Sci. USA, 98, 11857-11862.
17. Nguyen, J., Baldwin, M. A., Cohen, F. E. & Prusiner, S. B. (1995). Prion protein peptides induce alpha- helix to beta-sheet conformational transitions. Bio- chemistry, 34, 4186-4192.
18. Bouchard, M., Zurdo, J., Nettleton, E. J., Dobson, C. M. & Robinson, C. V. (2000). Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy. Protein Sci. 9, 1960-1967.
19. Fandrich, M., Fletcher, M. A. & Dobson, C. M. (2001). Amyloid fibrils from muscle myoglobin. Nature, 410, 165-166.
20. Klunk, W. E., Pettegrew, J. W. & Abraham, D. J. (1989). Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. J. Histochem. Cytochem. 37, 1273-1281.
21. Levine, H. (1995). Thioflavine-T interaction with amyloid ß-sheet structures. Amyloid, 2, 1-6.
22. Creighton, T. E. (1993). Proteins: Structures and Mol- ecular Properties, W. H. Freeman, New York.
23. Bothner, B., Lewis, W. S., DiGiammarino, E. L., Weber, J. D., Bothner, S. J. & Kriwacki, R. W. (2001). Defining the molecular basis of arf and hdm2 inter- actions. J. Mol. Biol. 314, 263-277.
24. Dobson, C. M. (1999). Protein misfolding, evolution and disease. Trends Biochem. Sci. 24, 329-332.
25. Pervushin, K., Riek, R., Wider, G. & Wuthrich, K. (1997). Attenuated T2 relaxation by mutual cancella- tion of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl Acad. Sci. 94, 12366-12371.
26. Sunde, M. & Blake, C. (1997). The structure of amy- loid fibrils by electron microscopy and X-ray diffrac- tion. Advan. Protein Chem. 50, 123-159.
27. Dobson, C. M. (2001). Protein folding and its links with human disease. Biochem. Soc. Symp., 1-26.
28. Lee, W., Harvey, T. S., Yin, Y., Yau, P., Litchfield, D. & Arrowsmith, C. H. (1994). Solution structure of the tetrameric minimum transforming domain of p53. Nature Struct. Biol. 1, 877-890.
29. Clore, G. M., Ernst, J., Clubb, R., Omichinski, J. G., Kennedy, W. M., Sakaguchi, K. et al. (1995). Refined solution structure of the oligomerization domain of the tumour suppressor p53. Nature Struct. Biol. 2, 321-333.
30. Jeffrey, P. D., Gorina, S. & Pavletich, N. P. (1995). Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms. Science, 267, 1498-1502.
31. Padrick, S. B. & Miranker, A. D. (2002). Islet amyloid: phase partitioning and secondary nucleation are cen- tral to the mechanism of fibrillogenesis. Biochemistry, 41, 4694-4703.
32. Morozova-Roche, L. A., Zurdo, J., Spencer, A., Noppe, W., Receveur, V., Archer, D. B. et al. (2000). Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants. J. Struct. Biol. 130, 339-351.
33. Fezoui, Y. & Teplow, D. B. (2002). Kinetic studies of amyloid beta-protein fibril assembly. Differential effects of alpha-helix stabilization. J. Biol. Chem. 277, 36948-36954.
34. Nielsen, L., Frokjaer, S., Brange, J., Uversky, V. N. & Fink, A. L. (2001). Probing the mechanism of insulin fibril formation with insulin mutants. Biochemistry, 40, 8397-8409.
35. Jiang, X., Buxbaum, J. N. & Kelly, J. W. (2001). The V122I cardiomyopathy variant of transthyretin increases the velocity of rate-limiting tetramer dis- sociation, resulting in accelerated amyloidosis. Proc. Natl Acad. Sci. USA, 98, 14943-14948.
36. Jones, S., Manning, J., Kad, N. M. & Radford, S. E. (2003). Amyloid-forming peptides from beta(2)- microglobulin-insights into the mechanism of fibril formation in vitro. J. Mol. Biol. 325, 249-257.
37. Myers, A. J. & Goate, A. M. (2001). The genetics of late-onset Alzheimer’s disease. Curr. Opin. Neurol. 14, 433-440.
38. Selkoe, D. J. (1999). Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature, 399, A23-A31.
39. White, J. T. & Kelly, J. W. (2001). Support for the mul- tigenic hypothesis of amyloidosis: the binding stoi- chiometry of retinol-binding protein, vitamin A, and thyroid hormone influences transthyretin amyloido- genicity in vitro. Proc. Natl Acad. Sci. USA, 98, 13019-13024.
40. Hashimoto, M., Hsu, L. J., Xia, Y., Takeda, A., Sisk, A., Sundsmo, M. & Masliah, E. (1999). Oxidative stress induces amyloid-like aggregate formation of NACP/alpha-synuclein in vitro. Neuroreport, 10, 717-721.
41. McLaurin, J., Yang, D., Yip, C. M. & Fraser, P. E. (2000). Review: modulating factors in amyloid-beta fibril formation. J. Struct. Biol. 130, 259-270.
42. Nielsen, L., Khurana, R., Coats, A., Frokjaer, S., Brange, J., Vyas, S. et al. (2001). Effect of environmen- tal factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemis- try, 40, 6036-6046.
43. Ratnaswamy, G., Koepf, E., Bekele, H., Yin, H. & Kelly, J. W. (1999). The amyloidogenicity of gelsolin is controlled by proteolysis and pH. Chem. Biol. 6, 293-304.
44. Zhu, M., Souillac, P. O., Ionescu-Zanetti, C., Carter, S. A. & Fink, A. L. (2002). Surface-catalyzed amyloid fibril formation. J. Biol. Chem. 277, 50914-50922.
45. Fabian, H., Mantsch, H. H. & Schultz, C. P. (1999). Two-dimensional IR correlation spectroscopy: sequential events in the unfolding process of the lambda cro-V55C repressor protein. Proc. Natl Acad. Sci. USA, 96, 13153-13158.
Edited by P. Wright
(Received 4 September 2002; received in revised form 23 January 2003; accepted 28 January 2003)