From the Center for Basic Neuroscience, Department of
Molecular Genetics, and Howard Hughes Medical Institute, and the
Departments of Biochemistry and Pharmacology, University of
Texas Southwestern Medical Center, Dallas, Texas 75390-9111, and the
§ Max-Planck-Institut für Biophysikalische Chemie,
37075 Göttingen, Germany
Received for publication, December 23, 2002, and in revised form, February 11, 2003
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ABSTRACT |
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In recent years, the presynaptic protein Production of Antibodies--
Antibodies were raised in rabbits against a
peptide corresponding to amino acid 2-24 of human Partial Tryptic Digestion--
NMR Spectroscopy--
NMR data were acquired at 25 °C on
Varian INOVA500 or INOVA600 NMR spectrometers. All NMR experiments were
performed with pulsed-field gradient enhanced pulse sequences (15, 16)
using H2O/D2O 95:5 as the solvent. Sample
conditions for 1H-15N HSQC spectra are
indicated in the corresponding figure legends. Backbone assignments
were obtained from three-dimensional
1H-15N NOESY-HSQC, TOCSY-HSQC, HNCO, HNCACB,
and CBCA(CO)NH experiments acquired on a 0.3 mM
sample of Miscellaneous--
SUVs of 100% brain phosphatidylcholine or
30% brain phosphatidylserine, 70% brain phosphatidylcholine (all from
Avanti Polar Lipids) were made and quantified as described (17). SUVs
were confirmed to be unilamellar by electron microscopy. SDS-PAGE and immunoblotting were performed using standard procedures. CD spectra were recorded at 25 °C in a Jasco Model J-720 instrument upgraded to
J-715U and equipped with a 6-position peltier cuvette holder. Recordings were made using Hellma Quartz cuvettes (path length 0.1 cm).
Spectra were averaged from 10-50 scans of 0.2 nm steps at a rate of 50 nm/min. Edman degradation was carried out using standard procedures.
Phospholipid-induced Folding of Detergent-induced
Because complexes of Folding Results in Partial Protection from
Proteolysis--
Limited tryptic digestion of NMR Analysis of Folded
The 1H-15N HSQC spectrum of Structural Analysis of Folded
The NOE data show that most of the 100 N-terminal residues, which
include the seven 11-residue repeats, adopt an
The lack of sequential dNN(i,i+1) and short-range
NOEs in the 40 C-terminal residues (Fig. 7) clearly shows that this
region of the molecule is largely unstructured. This conclusion is
further supported by the observation of sharper resonances in this
region than in the rest of the molecule (illustrated by the
1H-15N HSQC cross-peaks; Fig. 4D)
and by the small C
To assess the secondary structure of
The discovery of two Absence of Folding Intermediates--
At non-saturating
concentrations of SUVs or SDS, CD spectra of Implications for
The presence of two
Our results suggest that -Synuclein is a small cytosolic protein of
presynaptic nerve terminals composed of seven 11-residue repeats and a
hydrophilic tail.
-Synuclein misfolding and dysfunction may
contribute to the pathogenesis of Parkinson's disease and
neurodegenerative dementias, but its normal folding and function are
unknown. In solution,
-synuclein is natively unstructured but
assumes an
-helical conformation upon binding to phospholipid
membranes. We now show that this conformation of
-synuclein consists
of two
-helical regions that are interrupted by a short
break. The structural organization of the
-helices of
-synuclein was not anticipated by sequence analyses and may be
important for its pathogenic role.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-synuclein has
attracted much attention because of its involvement in
neurodegenerative diseases (1-3). Two independent mutations in human
-synuclein cause familial Parkinson's disease, and wild type
-synuclein is a major component of Lewy bodies, cytoplasmic
inclusion bodies found in Parkinson's disease and in several forms of
neurodegenerative dementia. However, independent of its role in
neurodegenerative diseases,
-synuclein is an interesting protein in
its own right. It is an abundant presynaptic protein that may regulate
neurotransmitter release and may contribute to synaptic plasticity
(4-6).
-Synuclein is the founding member of a protein family that
additionally includes
- and
-synucleins and synoretin (7-9). The
sequences of all synucleins are similar, although only
-synuclein is
implicated in disease. Synucleins are composed of six copies
(
-synuclein) or seven copies (all other synucleins) of an unusual
11-residue imperfect repeat, followed by a variable short hydrophilic
tail. Synucleins are soluble, natively unfolded proteins that avidly bind to negatively charged phospholipid membranes and become
-helical upon binding (10). Although secondary structure predictions indicate that the synuclein repeats could form an amphipathic structure
consistent with lipid binding, the
-helical conformation is puzzling
because the synuclein repeats are punctuated by central glycine
residues. Furthermore, in Lewy bodies
-synuclein is thought to be in
a
-strand aggregate, but aggregation of
-synuclein into dimers
and multimers is promoted by lipid environments that induce an
-helical conformation (11-13). In the present study, we have
examined the conformation of
-synuclein in lipidic environments to
understand the relation of its sequence to its physicochemical properties and to map a potential pathway of misfolding in
neurodegenerative disease.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-Synuclein--
Recombinant
-synuclein was
expressed in bacteria as GST-fusion proteins with a TEV protease
recognition sequence preceding the N-terminal methionine and cleaved
with TEV protease (Invitrogen), resulting in a single additional
glycine residue at the N terminus. After TEV cleavage,
-synuclein
was isolated as the only heat-stable component upon boiling for 15 min,
purified by ion-exchange chromatography on a MonoQ column (Amersham
Biosciences), and dialyzed against the appropriate experimental
buffers. N15 and C13/N15
double-labeled
-synuclein were prepared similarly from bacteria grown in minimal M9 media supplemented with N15
NH4Cl and C13D-Glucose (Cambridge
Isotope Laboratories).
-synuclein
(CDVFMKGLSKAKEGVVAAAEKTKQG) that was coupled via its N-terminal
cysteine to keyhole limpet hemocyanin, and against recombinant
-synuclein expressed in bacteria.
-Synuclein (13.8 µM) was incubated for the indicated times at room
temperature in the presence of
SUVs1 (280 molar excess;
composition: 30% phosphatidylserine/70% phosphatidylcholine or 100%
phosphatidylcholine) or SDS (1 mM) in 20 mM
Tris-HCl, pH 7.4, with 0.2% (SUVs) or 2% (SDS) trypsin (Sigma).
Digestion products were separated on 16.5% Tris-Tricine gels (14) and analyzed by Coomassie Blue staining or immunoblotting.
-synuclein dissolved in 20 mM phosphate
buffer, pH 7.4, plus 50 mM SDS. The 3D
1H-15N NOESY-HSQC data were also used to
analyze secondary structure NOE patterns and search for long-range NOEs.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-Synuclein--
Upon addition
of SUVs containing 30% phosphatidylserine and 70%
phosphatidylcholine, the CD spectrum of purified
-synuclein shifts
from a characteristic random coil pattern with a 195 nm minimum to a
typical
-helical pattern with 206 and 222 nm minima (Fig.
1A) (10). Thus
phosphatidylserine-containing SUVs induce
-synuclein folding
("folding" as used here refers to the adoption of a secondary, but
not necessarily tertiary structure). SUVs composed only of
phosphatidylcholine caused little change (data not shown). To determine
how many lipids are required for
-synuclein folding, we titrated
-synuclein with increasing amounts of SUVs with a precisely known
phospholipid content that were confirmed by electron microscopy to be
unilammellar. A progressive increase in
-synuclein folding was
observed, with apparent saturation of folding at a molar lipid/protein
ratio of ~270-300:1 (Fig. 1B). Taking into account that
SUVs are composed of phospholipid bilayers, this ratio indicates that
binding of each synuclein molecule requires ~140-150 phospholipid
molecules.
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Fig. 1.
Titration of
-synuclein folding with negatively charged
phospholipid vesicles. A, circular dichroism spectra of
-synuclein (13.8 µM) mixed with increasing
amounts of SUVs (composition: 30% brain phosphatidylserine, 70%
phosphatidylcholine; asterisk, scattering artifact at high
liposome concentrations). No changes were observed with pure
phosphatidylcholine SUVs (not shown). B, plot of the molar
ellipticity of
-synuclein at 222 nm as a function of the molar
lipid/protein ratio. Note that folding saturates at ~270-300
phospholipid molecules per
-synuclein molecule. Similar results were
obtained in three separate experiments.
-Synuclein Folding--
To examine the
conformation of folded
-synuclein, it is necessary to achieve
folding in solution. Therefore we used CD spectroscopy to examine a
series of detergents for their ability to support
-synuclein
folding. Most detergents tested (Nonidet P-40,
n-octylglycoside, Thesit, Tween 20, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid,
cholic acid, deoxycholic acid, and taurocholic acid) did not induce
folding (data not shown). Only lysosphatidylserine, lysophosphatidic
acid (but not lysophosphatidylcholine), and SDS elicited the same CD
change in
-synuclein as acidic SUVs (Fig. 2A and data not
shown).
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Fig. 2.
Folding of
-synuclein promoted by detergents.
A, circular dichroism spectra of
-synuclein in the
presence of SUVs containing phosphatidylserine or of SDS (13.8 µM
-synuclein was mixed with SUVs added at a molar
lipid to protein ratio of 280:1 or 1 mM SDS). B,
molar ellipticity of
-synuclein (13.8 µM) at 222 nm as
a function of SDS concentration (data shown are means ± S.E. of
three independent experiments).
-synuclein with SDS would be particularly
useful in characterizing its structure, we tested whether small
phospholipid vesicles and SDS induced similar folded states of
-synuclein. Titration of
-synuclein with increasing
concentrations of SDS revealed that SDS promotes folding below its
critical micellar concentration (7-10 mM), with complete
folding at 1 mM SDS for 13.8 µM
-synuclein
(Fig. 2B). This result suggests that ~70 SDS molecules are
required per
-synuclein molecule for complete folding, a number that
approximates the aggregation number for SDS (i.e. the number
of SDS molecules per micelle) (18). Thus it is possible that
-synuclein drives the formation of SDS micelles and folds, as in the
case of SUVs, on the surface of the micelles.
-synuclein revealed
that in the presence of saturating concentrations of SUVs containing
phosphatidylserine,
-synuclein was partly protected from
proteolysis, whereas SUVs lacking phosphatidylserine had no effect
(Fig. 3). Similarly, when we added 1 mM SDS, we also observed partial protection from proteolysis, although the precise pattern of protected fragments was
different, presumably because SDS affected the digestion. Immunoblotting with site-specific antibodies showed that the protected fragments (~6 kDa, ~4 kDa) under both conditions were derived from
the N-terminal region of
-synuclein (Fig. 3). We were unable to
ascertain the mass of these fragments by matrix-assisted laser desorption ionization time-of-flight experiments due to lipidic contaminants, but analysis by Edman degradation confirmed that the 4-6
kDa fragments correspond to the N terminus of
-synuclein. Judging
from their size, we surmised that one of the cleavage sites is probably
at lysines 43 or 45. In support of this conclusion, tryptic
digestion of mutant
-synuclein that lacks these lysines did not
produce 4-6 kDa protected fragments (data not shown).
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Fig. 3.
Partial tryptic digestion of
-synuclein. Digestion of
-synuclein (40 µg) by trypsin in the presence of SUVs composed of only
phosphatidylcholine (PC SUV) or of 30% phosphatidylserine,
70% phosphatidylcholine (PS SUV, both vesicles were added
at a molar lipid to protein ratio of 280:1), or in the presence of SDS
(1 mM). Digests were stopped at the indicated times, and
proteins were analyzed on Tris-Tricine gels stained with Coomassie Blue
(left panels). Edman degradation and parallel immunoblotting
with an antibody to the N-terminal 24 residues of
-synuclein
demonstrated that the protected fragments in the PS SUV and SDS samples
were derived from the N terminus of
-synuclein (right
panels). Numbers between the panels denote
migration of molecular weight markers.
-Synuclein--
To compare unfolded and
folded
-synuclein, we acquired 1H-15N HSQC
spectra of
-synuclein alone, or of
-synuclein in the presence of
either SUVs (30% brain phosphatidylserine/70% phosphatidylcholine) or
SDS (Fig. 4). The
1H-15N HSQC spectrum of
-synuclein alone
exhibited a dense cluster of cross-peaks over a narrow range,
confirming that the protein is unfolded as described previously (19).
The number of visible peaks (~50) was less than expected (135),
possibly because of aggregation or of fast chemical exchange between
amide groups and the solvent. After addition of saturating
concentrations of SUVs, many but not all of the
-synuclein
cross-peaks disappeared. This loss of cross-peaks presumably occurred
because the large size of the SUVs effectively broadened the resonances
of the corresponding amide groups of
-synuclein beyond detection.
The positions of
-synuclein cross-peaks that remained after binding
to SUVs were unchanged from the spectrum of
-synuclein alone (Fig.
4B), showing that these cross-peaks represent amino acids
that do not bind lipids and continue to be unfolded and mobile in the
presence of SUVs. Consistent with previous results (19), we observed 36 such amino acids that account for a quarter of the total sequence.
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Fig. 4.
NMR Spectra of
-synuclein in the absence and presence of SUVs or
SDS. A, 1H-15N HSQC
spectra of
-synuclein in 20 mM phosphate buffer, pH 7.4. B, overlay of the 1H-15N HSQC
spectra of
-synuclein in the absence and presence of 30% brain
phosphatidylserine, 70% phosphatidylcholine SUVs (0.1 mM
-synuclein with SUVs added at molar lipid to protein ratio of
280:1). Note the virtual superposition of the remaining peaks in the
SUV spectrum with those of the
-synuclein spectrum. These represent
amino acids that do not bind lipid and remain unfolded in the presence
of SUVs. C, overlay of the spectra of
-synuclein in
the absence and presence of SDS (20 mM). D,
high level view of the spectra of
-synuclein in the presence of SUVs
and SDS. Observe that most of the high intensity unfolded peaks in both
spectra coincide. Horizontal lines in A-D
indicate cross-peaks from Asn and Gln side chains.
-synuclein
obtained at saturating concentrations of SDS exhibited an increased
dispersion compared with that of isolated
-synuclein (Fig.
4C). This result is consistent with the formation of
-helical structure upon micelle binding revealed by CD spectroscopy
(Fig. 2). The number of visible cross-peaks also increased in SDS, most
likely because SDS disrupts aggregation and/or because the formation of
the
-helical conformation protected the amide groups from exchange
with the solvent. Interestingly, the strongest cross-peaks observed in
the presence of SDS were generally in the same positions as the
cross-peaks that remained in the 1H-15N HSQC
spectrum of
-synuclein bound to SUVs (Fig. 4D). Thus
these cross-peaks correspond to unfolded residues that do not
participate in binding to SDS micelles or to SUVs, supporting the
notion that the binding modes and folding of
-synuclein in both
environments are analogous.
-Synuclein--
To study the
structure of
-synuclein bound to SDS, we assigned its backbone
signals using triple resonance and three-dimensional 1H-15N NOESY-HSQC and TOCSY-HSQC experiments
(Fig. 5). Although a complete analysis of
short range NOEs that are diagnostic for different types of secondary
structure was made difficult by the strong resonance overlap observed,
numerous NOEs could be assigned unambiguously. These include multiple
NOEs that are characteristic of
-helical structure, particularly
dNN(i,i+1) and d
N(i,i+3)
NOEs, and are illustrated in sample strips from a three-dimensional
1H-15N NOESY-HSQC spectrum in Fig.
6. All dNN(i,i+1)
and d
N(i,i+3) NOEs that could be assigned are
summarized in Fig. 7 with black boxes and solid lines, respectively, whereas open
boxes and dashed lines indicate
dNN(i,i+1) and d
N(i,i+3)
NOEs that could not be assessed because of resonance overlap. Note that
dNN(i,i+1)/d
N(i,i+1) intensity ratios, rather than absolute dNN(i,i+1)
intensities, are represented in Fig. 7 to account for differences in
resonance line widths. Only a few d
N(i,i+2)
and d
N(i,i+4) NOEs could be assigned
unambiguously due to the resonance overlap and the limited sensitivity
of the data obtained under the conditions of these experiments
(data not shown).
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Fig. 5.
Backbone assignment of
-synuclein. Assignment of cross-peaks
in 1H-15N HSQC spectra of
-synuclein in the
presence of SDS (0.3 mM
-synuclein in 50 mM
SDS). Backbone signals were assigned using triple resonance and
three-dimensional 1H-15N NOESY-HSQC and
TOCSY-HSQC experiments. Resonance assignments have been deposited in
the BioMagResBank under accession number 5744.
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Fig. 6.
Composite of (F1, F3) strips of a
three-dimensional 1H-15N NOESY-HSQC spectrum of
SDS-bound -synuclein illustrating the NOE
patterns observed for residues 36-56. Solid lines
connect diagonal peaks with dNN(i,i+1) NOEs as
well as intraresidue d
N(i,i) NOEs with
d
N(i,i+3) connectivities.
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Fig. 7.
Secondary structure of
-synuclein bound to SDS micelles. The primary
sequence of
-synuclein is shown on top, and amide
protection data and diagnostic NOE patterns are summarized
below the sequence. Amide protection was assessed from the
intensities of exchange cross-peaks with the solvent in
three-dimensional 1H-15N NOESY-HSQC and
TOCSY-HSQC spectra. The intensities were classified in three categories
(weak, intermediate, and strong). Amide groups were considered
protected from exchange if no cross-peak or a weak cross-peak with the
solvent was observed (closed circles), whereas an
intermediate exchange cross-peak intensity indicated partial protection
(open circles). Question marks indicate amide
groups whose protection could not be assessed due to resonance overlap.
Solid boxes indicate the observation of
dNN(i,i+1) NOEs. The height of the boxes reflects
the intensity ratio between dNN(i,i+1) and
d
N(i,i+1) connectivities, classified into
three categories: >1, strong; 0.6-1, medium; <0.6, weak. Open
boxes indicate dNN(i,i+1) connectivities
that cannot be assessed due to resonance overlap. Observed
d
N(i,i+3) NOEs are represented by solid
lines, whereas dashed lines indicate
d
N(i,i+3) NOES that could not be assessed
because of resonance overlap.
-helical conformation. Interestingly, a noticeable break in the helical pattern
was observed around residues 43 and 44, revealing an interruption of
the helical structure in this region. This break in the helix was also
apparent from analysis of the deviations of the observed C
chemical
shifts from random coil values (Fig.
8A). Significant positive
deviations, which are characteristic of helical structure, were found
for most of the N-terminal 98 residues, but the C
chemical shifts of
residues 43 and 44 were close to random coil values. The abundance of
glycines in synuclein made analysis of H
chemical shift deviations
from random coil values difficult because the presence of two H
protons in each glycine introduces uncertainty in the corresponding
deviations. However, these data also illustrated the break in the helix
(Fig. 8B). This conclusion is based on the negative
deviations characteristic of helical structure that are observed for
most of the 100 N-terminal residues, whereas residues 43-45 exhibit
slightly positive deviations. In the sequences spanning residues
30-42, 60-65, and 82-100, a few H
deviations are close to zero,
and the C
deviations are relatively small compared with other parts
of the molecule, suggesting that there is helix fraying in these
residues. However, the C
deviations are still around 2 ppm or
larger, and these sequences exhibit abundant
dNN(i,i+1) and d
N(i,i+3)
NOE patterns. Thus, these sequences adopt predominantly
-helical
conformations that may be partially destabilized by the presence of
residues with low helical propensity such as glycines and
threonines.
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Fig. 8.
Conformational shifts of
-synuclein bound to SDS. Plots of the
differences between the C
(A) and H
(B)
chemical shifts observed for
-synuclein in SDS and those expected
for a random coil conformation as a function of the residue number.
Helical regions are characterized by positive values for
C
and
negative values for
H
. Random coil chemical shifts were
obtained from the BioMagResBank. The arrow identifies the
break in the
-helix at which the values for
C
and
H
resemble those of residues in a random coil
conformation.
and H
deviations from random coil chemical
shifts (Fig. 8; note that some significant deviations from random coil
values may be favored by the abundance of prolines in this region).
Extensive analysis of NOESY data did not uncover long-range NOEs
indicative of tertiary structure, suggesting that the two
-helices
do not interact with each other and are formed only at the
protein/micelle interface. This conclusion agrees with the moderate
chemical shift dispersion observed in SDS-bound
-synuclein, which
indicates the presence of a defined secondary structure but is lower
than the dispersion usually observed in globular proteins. Deuterium
exchange experiments monitored at pH 6.5 by
1H-15N HSQC spectra further reinforced the
conclusion that SDS-bound
-synuclein does not have a tertiary
structure because all amide groups were largely exchanged with the
solvent within the time scale of the first spectrum (30 min; data not shown).
-synuclein by an independent
approach, we examined the accessibility of each amino acid of
-synuclein to solvent by measuring their exchange cross-peaks with
water using three-dimensional 15N NOESY-HSQC and
TOCSY-HSQC spectra (summarized in Fig. 7). The intensity of the
exchange cross-peaks directly reflects the exposure of the respective
amino acid to solvent. We observed strong exchange cross-peaks, which
are indicative of a lack of amide protection for the unstructured
C-terminal residues consistent with the non-structured nature of the
corresponding sequence (see above). Furthermore, exchange cross-peaks
were weak or absent for much of the residues in the helical regions as
expected. In the region around residues 43-44, however, exchange
cross-peaks with intermediate intensity were observed, which confirms
the helical break in this region. Overall, the NMR data yield a picture
whereby the 100 N-terminal residues of
-synuclein bound to SDS do
not form a globular structure but adopt an
-helical conformation
with a break in the middle.
-helices in the folded conformation of
-synuclein was unanticipated as secondary structure calculations performed for example with the Expasy program suite
(us.expasy.org) predict a mixture of
-helices, random coils,
and
-strand. However, the NMR results correlate with the partial
proteolysis experiments described above, which uncovered N-terminal
protected 4 and 6 kDa bands that correspond to the first
-helix
(Fig. 3). The observation of this proteolysis pattern in
-synuclein
bound to both SUVs and SDS argues that their conformations are analogous.
-synuclein exhibited
intermediate molar ellipticities (Fig. 1). Under these conditions,
either only a subset of
-synuclein molecules was fully folded or a
defined region of all synuclein molecules, such as part of an
-helix, was folded before the other regions. The first model
implies that binding of both helical regions is a cooperative
all-or-none process, whereas the second model would involve
intermediate states containing partial helices. To distinguish between
these possibilities, we acquired an 1H-15N HSQC
spectrum of 0.1 mM synuclein at an intermediate SDS
concentration (2.8 mM). If partially helical intermediates
exist under these conditions, sharp cross-peaks in the positions
corresponding to the unbound protein would be expected for the regions
that remain unstructured. However, all of the cross-peaks corresponding
to the two
-helices that could be identified were substantially broadened, and their positions were close to those observed for fully
SDS-bound synuclein (data not shown). These results suggest that under
these conditions, synuclein is in an equilibrium between free and fully
bound states, although we cannot completely discard the possibility
that the bound states are somewhat different from those existing at
saturating SDS concentrations.
-Synuclein Function and Dysfunction--
We
have shown that SUVs containing phosphatidylserine and SDS induce
folding of
-synuclein in a concentration-dependent
manner, with saturation at ~140 phospholipid or 70 SDS molecules per
-synuclein molecule. In folded
-synuclein, the N-terminal 98 residues are structured, whereas the C-terminal 42 residues retain a
random coil configuration. The structured part of
-synuclein forms
two distinct
-helices that are interrupted by a two-residue break. Folding appears to be cooperative, without a stable intermediate at
non-saturating concentrations of SDS or SUVs. In the folded state, no
long-range interactions between
-synuclein residues were observed,
suggesting that there is no tertiary structure and that all of the
folded residues are directly or indirectly engaged in lipid binding.
-helices in
-synuclein and the position of
the break were unexpected.
-Synuclein is composed of seven imperfect
repeats composed of 11 residues. These repeats are interrupted after
the fourth repeat by a four-residue insertion. The break between the
two
-helices occurs in the first half of the fourth repeat, whereas
the four-residue inserted sequence is
-helical. Furthermore, the
break between the two
-helices consists of only two instead of four
residues (Fig. 9A). Thus the
break is not caused by the four-residue insertion between repeats;
rather, the four-residue insertion serves to maintain the helical
structure. When the two
-helices of
-synuclein are analyzed on a
helical net, an amphipathic distribution of residues emerges, which
explains the avid binding to negatively charged phospholipids (Fig.
9B). However, when the sequence of
-synuclein is modeled
into a single
-helix without a break, the orientation of the
hydrophobic surface shifts along the
-helix, suggesting that the
break between the two helices may allow a more favorable binding of
hydrophobic surfaces to the hydrophobic interior of the
micelle/membrane. Binding is likely to be stabilized by ionic
interactions between positive charged
-synuclein residues and
negatively charged phospholipid headgroups because no
-helicity was
observed with either membranes or detergents that do not contain
negative surface charges. An alternative explanation for the break is
that it may facilitate binding of
-synuclein to small vesicles with
a high curvature such as synaptic vesicles. If uninterrupted, the
-helix of synuclein would measure 14 nm, which may be too long to
coat a highly curved surface.
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Fig. 9.
Primary and secondary structure of
synucleins. A, sequence alignment of human synucleins
with identification of the eleven-residue repeats and the localization
of -helices. Residues shared among all synucleins are highlighted in
black, whereas residues shared among two of the four
synucleins are highlighted in gray. The two helices are
indicated by solid lines, with the break being denoted by
xx. The hatched lines below the alignment show
the eleven-residue repeats. B, two-dimensional
representation of the three-dimensional conformation of the N-terminal
98 residues of
-synuclein on a helical net.
-Synuclein is shown
in a modeled conformation as a single continuous
-helix without a
break (left) or in the observed conformation as two
-helices with a two-residue break (right). Hydrophobic
residues are shown in blue, charged residues in
red, and glycines in green. The N terminus with
the initiating methionine is at the bottom and the C
terminus on top as indicated.
-synuclein may function as a surface-active
coat of phospholipid membranes in the presynaptic nerve terminal,
possibly of synaptic vesicles (20, 21). Indeed, tryptic digestion of
-synuclein in the presence of synaptic vesicles resulted in
protection of N-terminal fragments similar to those seen with
phosphatidylserine-containing SUVs (data not shown), indicating that
-synuclein binds to and folds on synaptic vesicles. Alignment of the
four human synucleins shows that other synucleins probably adopt a
similar secondary structure as
-synuclein (Fig. 9A). The
sequences of the two
-helices and of the break are well conserved,
whereas the non-folded C termini are not. However,
-synuclein is
unique among synucleins because it has a longer second
-helix than
-synuclein, and a more hydrophobic surface than
-synuclein or
synoretin (Fig. 9A). These differences may explain the
selective propensity of
-synuclein to aggregate in neurodegenerative
diseases. Several recent studies (11-13) have shown that lipidic
environments that promote
-synuclein folding also accelerate
-synuclein aggregation, suggesting that the lipid-associated conformation described here may be relevant to
-synuclein misfolding in neurodegenerative diseases.
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ACKNOWLEDGEMENTS |
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We thank Dirk Fasshauer, Shigeo Takamori, and Wolfram Antonin for help and advice. We also thank Dietmar Riedel for analysis of SUVs by electron microscopy.
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FOOTNOTES |
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* This study was supported by grants from the National Institutes of Health (1-RO1-NS40057) (to T. C. S.) and the Deutsche Forschungsgemeinschaft (Gottfried-Wilhelm Leibnitz program) (to R. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence may be addressed. Tel.: 214-648-1876; Fax: 214-648-1879; E-mail: Thomas.Sudhof@UTSouthwestern.edu (to T. S.); or E-mail: Sreeganga.Chandra@UTSouthwestern.edu (to S. C.).
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M213128200
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ABBREVIATIONS |
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The abbreviations used are: SUV, small unilamellar vesicle; CD, circular dichroism; HSQC, heteronuclear single quantum correlation; NMR, nuclear magnetic resonance; NOE, nuclear Overhäuser effect; NOESY, nuclear Overhäuser Effect Spectroscopy; TOCSY, total correlation spectroscopy.
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