We fused fragments of Ab, residues 10–24 Ab1024 or 28–42 Ab2842, to three positions in the C-terminal region of ribonuclease HII from a hyper-thermophile, Thermococcus kodakaraensis Tk-R
Trang 12 PRESTO, Japan Science and Technology Agency (JST), Suita, Japan
3 Department of Applied Chemistry, Osaka University, Suita, Japan
Alzheimer’s disease (AD) is characterized by the
deposition of amyloid fibrils with a common cross
b-sheet structure [1] One component of the amyloid
deposits of Alzheimer’s disease has been identified as a
39–42 amino acid polypeptide, amyloid b peptide (Ab)
[2] Ab is derived from the proteolytic cleavage of the
amyloid precursor protein (APP), which is an integral
membrane protein [3]
Ab adopts a helix–turn–helix conformation in
2,2,2-trifluoroethanol (TFE) [4,5], SDS [6,7], and fluorinated
alcohol [8] However, the atomic-level crystal and solution structure of Ab in aqueous solution without organic solvents and detergents has not been deter-mined After proteolytic cleavage, Ab may remain sol-uble but eventually aggregate into a b sheet [1] The initial step of the process in which Ab undergoes a conformational transition from a soluble form to a
b structure in an aqueous environment remains unclear because conformational study of Ab in aqueous solu-tion is complicated by its tendency to aggregate
Keywords
Alzheimer’s disease; crystal structure;
fusion protein; conformational transition;
hyperthermophile protein
Correspondence
K Takano, Department of Material and Life
Science, Osaka University and PRESTO,
Japan Science and Technology Agency
(JST), 2-1 Yamadaoka, Suita, Osaka 565-0871,
Japan
Tel ⁄ Fax: +81 6 6879 4157
E-mail: ktakano@mls.eng.osaka-u.ac.jp
(Received 22 August 2005, revised 12
October 2005, accepted 7 November 2005)
doi:10.1111/j.1742-4658.2005.05051.x
Conformational studies on amyloid b peptide (Ab) in aqueous solution are complicated by its tendency to aggregate In this study, we determined the atomic-level structure of Ab28)42 in an aqueous environment We fused fragments of Ab, residues 10–24 (Ab10)24) or 28–42 (Ab28)42), to three positions in the C-terminal region of ribonuclease HII from a hyper-thermophile, Thermococcus kodakaraensis (Tk-RNase HII) We then exam-ined the structural properties in an aqueous environment The host protein, Tk-RNase HII, is highly stable and the C-terminal region has rel-atively little interaction with other parts CD spectroscopy and thermal denaturation experiments demonstrated that the guest amyloidogenic sequences did not affect the overall structure of the Tk-RNase HII Crys-tal structure analysis of Tk-RNase HII1)197–Ab28)42 revealed that Ab28)42 forms a b conformation, whereas the original structure in Tk-RNase HII1)213 was a helix, suggesting b-structure formation of Ab28)42 within full-length Ab in aqueous solution Ab28)42 enhanced aggregation of the host protein more strongly than Ab10)24 These results and other reports suggest that after proteolytic cleavage, the C-terminal region of Ab adopts
a b conformation in an aqueous environment and induces aggregation, and that the central region of Ab plays a critical role in fibril formation This study also indicates that this fusion technique is useful for obtaining structural information with atomic resolution for amyloidogenic peptides
in aqueous environments
Abbreviations
Ab, amyloid b peptide; AD, Alzheimer’s disease; AFM, atomic force microscope imaging; APP, amyloid precursor protein; FTIR, Fourier transform infrared; GdnHCl, guanidine hydrochloride; TFE, 2,2,2-trifluoroethanol; ThT, thioflavine T; Tk-RNase HII, ribonuclease HII from a hyperthermophile, Thermococcus kodakaraensis.
Trang 2Ab has been investigated using its fragments The
N-terminal region around residues 1–9 is not
import-ant for amyloid fibril formation [9] However, in the
central region, Ab16)22 comprises the central
hydro-phobic core that is thought to be important in
full-length Ab assembly [10–12] Ab11)25has been found to
form ordered amyloid fibrils, exhibiting similar
mor-phology to that of full-length Ab [13–15] Here, Abm–n
denotes residues m to n of Ab The C-terminal region,
including residues 28–42, is highly hydrophobic and is
important for nucleation in fiber formation [16,17]
We fused two sequences, residues 10–24 (Ab10)24)
and 28–42 (Ab28)42), of Ab (Fig 1A) to three
posi-tions in the C-terminal region of ribonuclease HII
from a hyperthermophile, Thermococcus kodakaraensis
(Tk-RNase HII) [18] in order to determine the
struc-tural properties of Ab sequences in aqueous solution
A schematic diagram of the designed variants is shown
in Fig 1B Tk-RNase HII is a stable protein with 228
amino acid residues [19] Because Tk-RNase HII is an
archaeal protein, it is not related to amyloid diseases
The crystal structure of Tk-RNase HII1)213 was
deter-mined and is shown in Fig 1C [20] Here, Tk-RNase
HIIm–n denotes residues m to n of Tk-RNase HII In
the structural analysis of Tk-RNase HII, a truncated
version of the protein (residues 1–213) has been
deter-mined [20] The C-terminal region (residues 197–212)
forms a helices but makes poor interaction with other
parts Because the fused proteins were not aggregated
in aqueous solution, we could examine their structural
properties, such as the circular dichroism (CD) spectra,
thermal stability and crystal structure We also
exam-ined the formation of aggregates of the mutant
pro-teins using thioflavine T (ThT)-binding analysis The
results confirmed the role of the regions in fibril
forma-tion of Ab in aqueous soluforma-tion
Results
Purification of recombinant Tk-RNase HII with
Ab fragment proteins Upon induction for overproduction, all the fusion pro-teins accumulated in the cells as a soluble form These fusion proteins were purified to give a single band on SDS⁄ PAGE by the same procedures used to purify the wild-type Tk-RNase HII These results suggest that the addition of Ab fragments does not significantly affect the three-dimensional structure of Tk-RNase HII under moderate conditions
Far-UV CD spectra
CD spectra of wild-type and mutant Tk-RNase HII were measured in the far-UV region to examine the effect of the Ab sequence on the overall secondary structure of Tk-RNase HII As shown in Fig 2, the shape of the spectra was almost the same for both wild-type and mutant proteins In particular, the value
of the negative peak at [h] 220 nm, which reflects the characteristic a-helix structure, did not change largely These results indicate that a large transformation from a- to b-conformation was not observed and that the overall structure of the Tk-RNase HII was not dra-matically changed by the fused Ab fragments in the C-terminal region
Although the secondary structures at the C-terminal region would be locally changed or deleted in mutant proteins, CD spectra of the mutant proteins were sim-ilar to that of the wild-type protein This may be that
CD signal of Tk-RNase HII is mainly from the other eight a helices
A
B
C
Fig 1 (A) Sequence of amyloid b peptide
(Ab) The regions with underbars correspond
to Ab10)24and Ab28)42 (B) Schematic
dia-gram of the wild-type and designed variants
for Tk-RNase HII (C) Crystal structure of
ribonuclease HII from a hyperthermophile,
Thermococcus kodakaraerisis (Tk-RNase HII)
[19] Blue represents the C-terminal helix
(residues 197–212) The structures were
drawn using the program RASMOL
Trang 3Thermal stability
We measured heat stability to investigate the changes
in conformational stability of the Tk-RNase HII
vari-ants due to the addition of Ab fragments Because
Tk-RNase HII is very stable against heat-induced
dena-turation [19], we added 1.2 m GdnHCl to the solution
The heat-induced denaturation was highly reversible It
has been reported that the heat-induced unfolding of
Tk-RNase HII does not attain equilibrium at this scan
rate because of its remarkably slow unfolding [19]
Therefore, in this study, we estimated the Tmvalue for
the wild-type and mutant Tk-RNase HII proteins, as
listed in Table 1 The Tmvalue changed from +2.0 to
)2.0 C after exchange of Ab fragments at the
C-ter-minal region These results show that fused Ab
frag-ments do not seriously affect the thermal stability of
Tk-RNase HII
Crystal structure
For Tk-RNase HII1)197–Ab28)42, orthorhombic
crys-tals appeared under one of the crystallization
condi-HII1)197–Ab28)42 diffracted to 2.8 A˚ at a synchrotron source This crystal belongs to the space group P21212, having one protein molecule per asymmetric unit The structure of the protein was analyzed using the molecular replacement method with the wild-type Tk-RNase HII1)213 structure [20] as a probe molecule The root mean square deviations from the ideal bond length and the angle were 0.008 A˚ and 1.190 Analysis
of the stereochemistry with the program procheck [21] demonstrated that 89.5% of the nonglycine resi-dues were in the most favorable region of the Rama-chandran plot; 10.5% were in the additionally allowed region, and no residues were in the disallowed region The overall structure of Tk-RNase HII1)197–Ab28)42
is similar to the wild-type except for the regions sur-rounding the C-terminus, as shown in Fig 3A Resi-dues 70–80 near the C-terminus changed the structure from a small a helix to a loop, and the C-terminal helix where Ab28)42 was introduced was converted to a
b structure (see below) The other parts, however, have almost the same conformations as the wild-type struc-ture This result confirms the small effect of Ab frag-ments on the overall structure of Tk-RNase HII The electron density around the C-terminus is depic-ted in Fig 3B The electron density shows that this region, which is a helix in the wild-type structure, does not form an a-helix conformation The model based
on electron density indicates that the helical conforma-tion at the C-terminus changes into a small antiparallel
b sheet (Fig 3C) The Ab28)42 fragment introduced forms a b conformation even if the original structure was an a conformation (Fig 3D) This suggests that the Ab28)42 region in Ab1)42 also retains b conforma-tion in aqueous soluconforma-tion
The new b sheet interacts with the a helix (residues 197–212) The side chain of Glu181 in the a helix hydrogen bonds with the main chain of Leu204 (Ab– Leu34) The side chains of Ile201, Val210 and Ile211 (Ab–Ile31, Val40 and Ile41) make a hydrophobic core within the molecule These interactions may stabilize the b sheet, but the b sheet has relatively little inter-action with other parts
Formation of aggregates
We measured the formation of aggregates of the wild-type and variant proteins using ThT-binding analysis
in order to determine whether the introduced amyloid-ogenic sequences induce amyloid formation in the host protein Under artificial conditions, 10% TFE at
70C, the structure of the wild-type protein was
Fig 2 CD spectra of the wild-type and six mutants for Tk-RNase
HII The red line represents the wild-type protein Black lines
repre-sent the mutant proteins The protein concentration was
0.14 mgÆmL)1in 20 m M Tris ⁄ HCl at pH 8 and 25 C.
Table 1 Tm value for the wild-type and mutant Tk-RNase HII
pro-teins (60 C ⁄ h in 20 m M Tris ⁄ HCl, 1.2 M GdnHCl at pH 8).
Tm(C)
Trang 4changed, detected by CD spectra (data not shown).
Aggregation, however, did not occur, even after
four days Figure 4 illustrates the results Like
wild-type Tk-RNase HII, Tk-RNase HII1)197–Ab10)24 and
Tk-RNase HII1)174–Ab10)24 did not aggregate By
contrast, Tk-RNase HII1)212–Ab28)42 and Tk-RNase
HII1)174–Ab28)42 aggregated rapidly Although only
Tk-RNase HII1)212–Ab10)24exhibited the formation of
aggregates among the variants with Ab10)24, the
ten-dency of Tk-RNase HII1)212–Ab10)24 was lower than
that of Tk-RNase HII1)212–Ab28)42 We conclude that
Ab28)42 enhances the aggregation of the host protein
more strongly than Ab10)24
Discussion
Effects of Ab fragment attachments on the
structure of a hyperthermophile protein
In this study, we added Ab sequences to the
C-ter-minal regions of Tk-RNase HII, which is the stable
protein from a hyperthermophile [19] The
overexpres-sion system of Tk-RNase HII using Escherichia coli has been constructed It takes a monomer form in aqueous solution [18] The crystal structure was deter-mined, and it was found that the C-terminal region interacts relatively little with other parts of the mole-cule [20] Thus, it is thought that Tk-RNase HII is a good model as a host protein for analyzing the struc-tural properties of amyloidogenic peptides in aqueous environments
Protein purification, CD spectra and thermal dena-turation experiments on variant proteins with Ab sequences revealed that the overall native structure of Tk-RNase HII was not susceptible to the exchange of
Ab sequences in the C-terminal regions, even after 15 residues were replaced and up to 39 residues were dele-ted This is thought to be because Tk-RNase HII is too robust to modify the overall structural properties and the C-terminal region of Tk-RNase HII is vari-able
Although the introduced Ab sequences do not seri-ously affect the overall native conformation, some of them induce aggregation of the host protein under
Fig 3 (A) Overall structures of Tk-RNase HII1)197–Ab28)42and wild-type Tk-RNase HII (Tk-RNase HII1)213) Blue (yellow) lines represent the wild-type (mutant) proteins (B) Electron density around the C-terminal region of Tk-RNase HII1)197–Ab28)42 A 2 Fo–Fcmap contoured at 1.2 o´ is shown (C) C-Terminal region of Tk-RNase HII1)197–Ab28)42 The images were prepared using O The red dotted lines represent the hydrogen bonds (D) Overall structures of wild-type Tk-RNase HII (Tk-RNase HII1)213) (left) and Tk-RNase HII1)197–Ab28)42(right) Blue repre-sents the C-terminal region The structures were drawn using the program RASMOL
Trang 5artificial conditions It is well known that proteins with
no relevance to amyloid disease sometimes form
amy-loid fibrils under artificial conditions in which they are
partially denatured [22–24] In this case, some variants
of Tk-RNase HII aggregated in 10% TFE solution at
70C, although the wild-type Tk-RNase HII did not
Because the stability of these variants is high, the
results represent the possibility of amyloid fibril
forma-tion by even stable proteins Yutani et al [25] also
report amyloid-like fibril formation by methionine
aminopeptidase from a hyperthermophile, Pyrococcus
furiosus, in the presence of 3.37 m GdnHCl at pH 3.3
Although it has been reported that the formation of
aggregates depends on protein stability [26–29], there
was no correlation between stability and the formation
of aggregates among the proteins in this study,
indica-ting the sequence dependence of amyloidogenicity in
the case of Ab
Tk-RNase HII1)212–Ab28)42and Tk-RNase HII1)174–
Ab28)42 increased rapidly in fluorescence intensity
under ThT-binding analysis We observed only
depo-sits, and not amyloid fibrils, of these proteins using
atomic force microscope imaging (AFM) (unpublished
data) Because these proteins bound ThT, the deposits
might be proto-filament or short fibrils The results
indicate that only Ab28)42 enhances aggregation of the
Ab29)42 both form b sheet, but Ab29)42 forms only very short fibrils, whereas Ab10)23 forms long fibrils [15] Furthermore, Ab16)22 comprises the central hydrophobic core that is thought to be important in full-length Ab assembly [10–12] Ab11)25 has been found to form ordered amyloid fibrils, exhibiting a morphology similar to that of full-length Ab [13–15] The study of hydrogen–deuterium exchange mapping
of Ab using NMR revealed that the middle of Ab is involved in a b structure in the fibrils [30] In contrast, the C-terminal region, including residues 28–42, is important in nucleation for fiber formation [16,17] These results coincide with our results and suggest that the C-terminal region of Ab induces aggregation but not elongation of fibrils, and that the central region of
Ab plays a critical role in fibril formation In the case
of Tk-RNase HIIm–n–Ab28)42, the proteins move up to aggregation but not to fibrils because of the lack of Ab central region In the case of Tk-RNase HIIm–n–
Ab10)24, the proteins are not aggregated efficiently, because of the absence of an Ab C-terminal region
Conformation of Ab28)42in aqueous environments
Structural studies of amyloid fibrils from Ab have been performed using CD [30], X-ray fiber diffraction [32], electron microscopy [14], and solid-state NMR [15], and the fibrils have been well characterized However, the mechanism of the conformational transition from a-helical conformation into b structure has not yet been fully understood because the atomic-level conforma-tions of Ab in aqueous environments are unclear Fur-thermore, there are some reports that soluble forms of
Ab possess intrinsic neurotoxicity [33,34], indicating the importance of structural analysis in aqueous solutions
In APP, Ab1)28 is in the extracellular domain and
Ab29)42 is in the transmembrane domain [35] Struc-tural analysis of Ab in membrane-mimicking solvents indicates that Ab28)42 takes an a-helix form [7], sug-gesting likely a-helix formation of Ab28)42 in the mem-brane environment of APP After proteolytic cleavage, soluble nonfibrillar forms of Ab exist in the extracellu-lar medium in vivo, whereas Ab is known to incorpor-ate into amyloid fibrils [36] In this study, we resolved the crystal structure of Ab28)42 using fusion proteins and discovered the b-sheet formation of Ab28)42 in an aqueous environment This is the first report of the atomic-level structure of Ab fragments in aqueous solution The present result coincides with results from
CD spectroscopy and Fourier-transform infrared
Fig 4 Thioflavine T-binding analysis of the wild-type and six
mu-tants for Tk-RNase HII Wild-type (red cross), Tk-RNase HII 1 )212–
Ab10)24(solid squares), Tk-RNase HII1)212–Ab28)42(open squares),
Tk-RNase HII1)197–Ab10)24 (solid circles), Tk-RNase HII1)197–
Ab 28 )42 (open circles), Tk-RNase HII1 )174–Ab10 )24 (solid triangles)
and Tk-RNase HII1)174–Ab28)42 (open triangles) The protein
solu-tion (0.5 mgÆmL)1), in 20 m M NaOH ⁄ Cit (pH 3) with 10% TFE, was
incubated at 70 C for 1, 2, 3, and 4 days 20 lL of each protein
solution was added to 980 lL of 5 l M ThT in 50 m M glycine ⁄ NaOH
at pH 8.5 Fluorescence intensity was measured at 446 nm
excita-tion and 482 nm emission at 25 C.
Trang 6(FTIR) spectroscopy [9], and with the structural
pre-diction of Ab28)42 [37] Thus, we confirm the b
confor-mation of the region 28–42 in soluble nonfibrillar
forms of Ab This study also demonstrates the ability
to resolve the atomic-resolution structure of Ab
frag-ments using this host–guest technique with a stable
host protein, in spite of their aggregative nature in
aqueous environments Further studies will reveal the
conformations of other Ab fragments, including
full-length Ab
The two most abundant forms of Ab are the 40
and 42 residue peptides, Ab1)40 and Ab1)42 Both
forms are capable of assembling into b-sheet fibrils
However, Ab1)42 plays an important role in the
path-ogenesis of AD The formation of aggregates and
neurotoxicity of Ab1)42 are higher than those of
Ab1)40 [38] Until now, there has been no persuasive
explanation regarding this issue This study found
antiparallel b-sheet formation in the C-terminal
region If Ile41 and Ala42 are absent, it is difficult to
form a stout antiparallel b sheet within the molecule
Therefore, the two C-terminal residues are important
for antiparallel b-sheet formation of soluble
nonfibril-lar forms of Ab, which would nucleate and induce
overall b-sheet fibrils It has been reported that I41T
and A42T of Ab1)42 have aggregative potential like
Ab1)42, whereas V40P, I41P and A42P hardly
aggre-gate [39], supporting the b-sheet hypothesis for the
C-terminal region in Ab
Experimental procedures
Protein purification
The plasmids for overexpression of the mutant Tk-RNase
HII were constructed from those of the wild-type Tk-RNase
HII using standard recombinant DNA techniques
Over-production and purification of all proteins were performed
as reported for the wild-type protein [18] The purity of the
proteins was confirmed using SDS⁄ PAGE The
concentra-tion of the wild-type was estimated by assuming A280nm of
0.63 for a 1 mgÆmL)1protein [20] The concentration of each
variant was corrected using the Trp and Tyr content [40]
CD spectroscopy
CD measurements in the far-UV region were carried out on
a J-725 automatic spectropolarimeter (Japan Spectroscopic
Co., Ltd, Hachioji, Japan) The concentration of the
pro-teins was 0.14 mgÆmL)1in 20 mm Tris⁄ HCl at pH 8 and a
temperature of 25C Cells with path length of 2 mm were
used The mean residue ellipticity, h, which has units of deg
cm2Ædmol)1, was calculated
Thermal denaturation measurement Thermal denaturation curves for the wild-type and variant Tk-RNase HII were determined by measuring the change in
CD at 220 nm in 0.14 mgÆmL)1in 20 mm Tris⁄ HCl, 1.2 m GdnHCl at pH 8, in 2 mm cuvettes The measurement was made on a J-725 automatic spectropolarimeter The heating rate was 60CÆh)1 The curves were analyzed by a nonlinear least-squares analysis [41] using the following equation:
y¼ ððyfþ mf½TÞ þ expððDHm=RTÞððT TmÞ=ðTmÞÞ
ðyuþ mu½TÞÞ=ðð1 þ expðDHm=RTÞððT TmÞ=ðTmÞÞÞ ð1Þ Here, yf+ mf [T] and yu+ mu [T] describe the linear dependence of the pre- and post-transitional baselines on temperature, DHm is the enthalpy of denaturation at Tm, and Tm is the midpoint of the thermal denaturation curve Curve fitting was performed using microcal origin curve-fitting software (MicroCal, Northampton, MA)
Crystallization The crystallization condition of the mutants of Tk-RNase HII was initially screened using crystallization kits from Hampton Research (Alise Viejo, CA, USA) (Crystal Screens I, II, Cryo and Light), Emerald Biostructures (Bainbridge Island, WA, USA) (Wizard I, II, Cryo I and Cryo II), and Molecular Dimensions (Apopka, FL, USA) (Stura Footprint) with a semiautomatic protein crystalliza-tion system, TASCAL-1 (Kentoku Industry Co., Ltd, Sulta, Japan) [42] The crystallization condition was surveyed using the sitting-drop vapor-diffusion method at 20C Drops were prepared by mixing 1 lL each of the protein solution (14 mgÆmL)1) and the reservoir solution The drops were then vapor-equilibrated against 100 lL of the reservoir solution using 96-well Corning CrystalEX Microplates (Hampton Research) Single crystals of TkRNase -HII1)197–Ab28)42 appeared after five days using 200 mm Mes, 11% PEG8000, and 20% Glycerol at pH 7 The cry-stallization conditions were further optimized, resulting in the appearance of single crystals suitable for X-ray diffrac-tion analysis, when a microstirring technique [43] on Combi-Clover Plates (B-Bridge, Sunnyvale, CA, USA) was used
X-Ray diffraction and refinement
A crystal of Tk-RNase HII1)197–Ab28)42was mounted on a CryoLoop (Hampton Research) and then flash-frozen in a nitrogen stream at 100 K X-Ray diffraction data on the BL38B1 were collected at SPring-8, Japan, using a Quan-tum 4R CCD detector (Area Detector System Corp., Poway, CA, USA) A total of 180 images were recorded with an exposure time of 5 s per image and an oscillation angle of 1 Processing of the diffraction images and scaling
of the integrated intensities were performed using the
Trang 7program hkl2000 [44] The data collection statistics are
shown in Table 2
To determine the structure of Tk-RNase HII1)197–
Ab28)42, molecular replacement was performed with amore
[45], using chain A of the crystal structure of the wild-type
Tk-RNase HII (Tk-RNase HII1)213, PDB entry 1IO2) as a
search model Model-building and refinement of the
struc-ture was accomplished using the programs o [46] and cns
[47] The refinement statistics are presented in Table 1 The
coordinates of Tk-RNase HII1)197–Ab28)42have been
depos-ited in the Protein Data Bank under accession code 1·1P
ThT-binding analysis
Amyloid quantification in solution was performed using the
ThT method [48] The protein solution (0.5 mgÆmL)1), in
20 mm NaOH⁄ Cit (pH 3) with 10% TFE, was incubated at
70C for 1, 2, 3, and 4 days Twenty microliters of each
protein solution was added to 980 lL of 5 lM ThT in
50 mm glycine⁄ NaOH at pH 8.5 Fluorescence intensity
was measured at 446 nm excitation and 482 nm emission
using a Hitachi F2000 spectrofluorometer (Tokyo, Japan)
Acknowledgements
This work was supported in part by a Grant-in-Aid
for National Project on Protein Structural and
Culture, Sports, Science, and Technology of Japan, and by an Industrial Technology Research Grant Program from the New Energy and Industrial Tech-nology Development Organization (NEDO) of Japan The synchrotron radiation experiments were per-formed at the BL38B1 in the SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal no 2004A0680-NL1-np-P3k)
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