J._Biol._Chem.-2012-Bugg-31739-46.pdf

Structural Features and Domain Organization of

Huntingtin Fibrils

□

Received for publication, February 16, 2012, and in revised form, July 7, 2012

Published, JBC Papers in Press, July 16, 2012, DOI 10.1074/jbc.M112.353839

Charles W. Bugg

‡1,2

, J. Mario Isas

§1

, Torsten Fischer

, Paul H. Patterson

‡

, and Ralf Langen

§3

From the

‡

Biology Division, California Institute of Technology, Pasadena, California 91125 and the

Department of Biochemistry

and Molecular Biology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California,

Los Angeles, California 90089-2821

Background:

The structure of fibrils formed in Huntington disease remains unknown.

Results:

Local structure and domain organization of huntingtin exon 1 fibrils were determined by EPR spectroscopy.

Conclusion:

In contrast to the C terminus, the N terminus, and polyglutamine core regions become closely packed, without

forming a parallel, in-register structure.

Significance:

The results provide structural constraints and insight into how adjacent domains affect polyglutamine

structure.

Misfolding and aggregation of huntingtin is one of the hall-

marks of Huntington disease, but the overall structure of these

aggregates and the mechanisms by which huntingtin misfolds

remain poorly understood. Here we used site-directed spin

labeling and electron paramagnetic resonance (EPR) spectros-

copy to study the structural features of huntingtin exon 1

(HDx1) containing 46 glutamine residues in its polyglutamine

(polyQ) region. Despite some residual structuring in the N ter-

minus, we find that soluble HDx1 is highly dynamic. Upon

aggregation, the polyQ domain becomes strongly immobilized

indicating significant tertiary or quaternary packing interac-

tions. Analysis of spin-spin interactions does not show the close

contact between same residues that is characteristic of the par-

allel, in-register structure commonly found in amyloids. Never-

theless, the same residues are still within 20 Å of each other,

suggesting that polyQ domains from different molecules come

into proximity in the fibrils. The N terminus has previously been

found to take up a helical structure in fibrils. We find that this

domain not only becomes structured, but that it also engages in

tertiary or quaternary packing interactions. The existence of

spin-spin interactions in this region suggests that such contacts

could be made between N-terminal domains from different

molecules. In contrast, the C-terminal domain is dynamic, con-

tains polyproline II structure, and lacks pronounced packing

interactions. This region must be facing away from the core of

the fibrils. Collectively, these data provide new constraints for

building structural models of HDx1 fibrils.

Huntington disease (HD)

is a progressive, fatal neurodegen-

erative disorder caused by a polyglutamine (polyQ) expansion

in the first exon of the huntingtin (Htt) (1). Like other polyQ

diseases (2), there is a threshold of about 40 Gln beyond which

patients get the disease, and the age of onset of the disease is

inversely correlated with the length of the expansion (3). HD is

associated with neurodegeneration, especially of the caudate

nucleus of the striatum (4), and the severity of neurodegenera-

tion correlates with polyQ length (5). Huntingtin exon 1

(HDx1), with a polyQ expansion beyond a threshold of about 40

Gln, is sufficient to cause disease in mice (6).

A hallmark of HD in mice and humans is the formation of

intracellular inclusions. In patients, these inclusions consist

largely of N-terminal fragments of mutant Htt that are often

ubiquitinated (7, 8). Aggregates purified from HD patient

brains bind Congo Red with green birefringence, indicating

that they have an amyloid-like structure (9).

In vitro

, mutant

HDx1 spontaneously forms fibrils that similarly bind Congo

Red (9). Fourier transform infrared spectrometry demonstrated

that these fibrils have a

-sheet signature similar to amyloid

fibrils (10). Furthermore, HDx1 fibrils have an x-ray diffraction

pattern consistent with the cross-

structure characteristic of

amyloid fibrils (11, 12). In the cross-

structure,

-strands sep-

arated by 4.8 Å run normal to the fibril axis and form a sheet

that is parallel to the fibril axis.

HDx1 consists of three domains, an N-terminal 17 amino

acids, a polyQ region of variable length, and a C terminus that is

rich in prolines. There are several lines of evidence indicating

that the polyQ region of HDx1 forms the amyloid core. For

example, studies of synthetic polyQ peptides indicate that

aggregation propensity increases with polyQ length (13), and

that amyloid-like aggregates are formed (14). The polyQ flank-

ing regions in HDx1 further modulate its aggregation and tox-

icity (15–20). Interestingly, a recent solid-state NMR study

using a fragment of HDx1 indicates that the N terminus can

This work was supported, in whole or in part, by a grant from the National

Institutes of Health through the NINDS (to P. H. P.). This work was also sup-

ported by grants from the Hereditary Disease Foundation (to R. L. and

P. H. P.).

□

This article contains

supplemental Figs. S1 and S2

Both authors contributed equally to this work.

Present address: Keck School of Medicine, University of Southern California,

Health Sciences Campus, Los Angeles, CA 90089.

To whom correspondence should be addressed: Dept. of Biochemistry and

Molecular Biology, Zilkha Neurogenetic Institute, Keck School of Medicine

at the University of Southern California, 1501 San Pablo St., ZNI 119, Los

Angeles, CA 90089-2821. Tel.: 323-442-1323; Fax: 323-442-4404; E-mail:

langen@usc.edu.

The abbreviations used are: HD, Huntington disease; HDx1, huntingtin

exon 1; Htt, huntingtin; MTSL,

-(2,2,5,5-tetramethyl-2,5-dihydro-1H-

pyrrol-3-yl)methyl methanesulfonothioate; polyQ, polyglutamine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 38, pp. 31739–31746, September 14, 2012

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adopt an

-helical structure in the fibril (21). The proline-rich

C terminus is thought to retard fibril formation by preventing

the transition of the polyQ region to an amyloid-like structure

(22). Aside from the assignment of helical secondary structure

to the N terminus and a cross-

structure to the polyQ region,

little is known about the molecular organization of HDx1

fibrils. Moreover, previous studies employed the smaller frag-

ments of HDx1 rather than the entire HDx1 protein. As a con-

sequence, the structural features are even less understood in the

context of the entire HDx1 protein.

Here we present electron paramagnetic resonance (EPR)

results characterizing the domain organization of recombi-

nantly made, full-length HDx1 fibrils. We show that although

much of the polyQ region is immobilized in the fibril, it does not

have the parallel, in-register signature characteristic of many

other disease-causing amyloid structures. The C terminus

exhibits the greatest mobility, takes up polyproline II structure,

and loosens the packing of adjacent polyQ residues. Finally, the

N terminus is ordered and, in contrast to the C terminus, makes

tertiary or quaternary contact with other N termini, leading to

stabilization of the fibril core.

EXPERIMENTAL PROCEDURES

Protein Expression, Labeling, and Purification

—Using site-

directed mutagenesis, both thioredoxin cysteines were mutated

to serines in pET32a-HD46Q to create a parent construct for

making cysteine mutants of Htt. Cysteine mutations were

introduced by site-directed mutagenesis into the parent con-

struct, which expresses a thioredoxin fused to the N terminus of

Htt that has 46 glutamines and a C-terminal His tag (NTRX-

Q46). Overnight cultures of BL21(DE3) were diluted 50-fold

into LB medium and grown at 37 °C to 0.6

600

. Isopropyl

1-thio-

-galactopyranoside was added to 1 m

, and the tem-

perature was reduced to 30 °C for 4 h. Pellets were collected by

centrifugation at 3500

, resuspended in 20 m

Tris-HCl, pH

8.0, 300 m

NaCl, and 10 m

imidazole containing 1

CelLytic

B Cell Lysis reagent (Sigma-Aldrich), and incubated for 20 min

at room temperature on a rocker. Lysates were clarified by cen-

trifugation at 21,000

for 10 min and incubated with nickel-

nitrilotriacetic acid-agarose beads (Qiagen) fo

r1hat4°Cona

rocker. Beads were decanted into an Econo-Pac chromatogra-

phy column (Bio-Rad) and washed with several column vol-

umes of 20 m

Tris-HCl, pH 8.0, 300 m

NaCl, 20 m

imidaz-

ole. Purified proteins were eluted with 20 m

Tris-HCl, pH 8.0,

300 m

NaCl, 250 m

imidazole. Volumes were reduced to

250

l using Amicon Ultra-4 or Ultra-15 3000 MWCO cen-

trifugal filters (Millipore), after which the protein was incu-

bated with an equal volume of immobilized TCEP disulfide

reducing gel (Pierce) for1hatroom temperature. Following

reduction, the disulfides were spin-labeled by incubation with a

5–15-fold excess of MTSL spin label (Toronto Research Chem-

icals, Inc., North York, ON, Canada) fo

r1hatroom tempera-

ture. Labeled protein was FPLC-purified on a Superdex-75 gel-

filtration column (GE Healthcare) using phosphate-buffered

saline (137 m

NaCl, 2.7 m

KCl, 10 m

HPO

, 1.76 m

NaH

), adjusted to pH 6.8 with phosphoric acid, containing

EDTA. For dilution spectra, unlabeled fusion protein

without cysteines was purified in the same manner, except no

MTSL was added. Unlabeled fusion protein without cysteines

used to make seeds was purified similarly, except during FPLC,

50 m

Tris-HCl, pH 8.0, 150 m

NaCl, 1 m

EDTA were used.

Preparation of Seeds

—Unlabeled fusion protein without cys-

teines in 50 m

Tris-HCl, pH 8.0, 150 m

NaCl, 1 m

EDTA

was diluted to 5

(225

g/ml). To cleave the thioredoxin tag

and initiate fibril formation, EKMax (Invitrogen) was added to

1 unit/10

g of NTRX-Q46. The reaction was incubated with-

out agitation at 4 °C for 3 days. Fibril formation appeared to be

complete by electron microscopy, as judged by the absence

globular species. Fibrils were collected by ultracentrifugation at

150,000

for 20 min and resuspended in one tenth of the

original volume. To fragment the fibrils, this suspension was

then sonicated on maximum power for 10 min at 30-s intervals.

Sonicated seeds were stored at

80 °C.

Fibril Formation

—All Htt protein concentrations were

measured by BCA assay and adjusted to 225

g/ml (5

) for

fibril formation. For mobility measurements, reactions were set

up using 10% labeled mutant protein and 90% unlabeled protein

without cysteines. To measure spin-spin interactions, reactions

containing 100% labeled protein were used. To minimize

batch-to-batch variation, all reactions were seeded with 10%

preaggregated seed fibrils, and fibril formation was initiated by

the addition of 1 unit of EKMax/10

g of protein. Reactions

were incubated overnight at 4 °C without agitation. Fibrils were

collected by ultracentrifugation at 150,000

for 20 min and

resuspended in 7

l of PBS, pH 6.8, with 1 m

EDTA.

Electron Microscopy

—Prior to ultracentrifugation, 6

lof

fibrils was removed and adsorbed onto copper mesh electron

microscopy grids (Electron Microscopy Sciences, Hatfield, PA)

for 2 min. These grids were negatively stained with 2% uranyl

acetate for 2 min. Subsequently, the grids were examined with a

JEOL JEM-1400 electron microscope (JEOL, Peabody, MA) at

100 kV and photographed using a Gatan digital camera.

Continuous Wave EPR Spectra

—After ultracentrifugation,

the resuspended fibrils were loaded into quartz capillaries

(0.6-mm inner diameter

0.84-mm outer diameter, Vitro-

Com, Mt. Lakes, NJ), and EPR spectra were recorded on an

X-band Bruker EMX spectrometer (Bruker Biospin Corpora-

tion) at room temperature. The scan width was 150 gauss using

a HS cavity at an incident microwave power of 12.60 milliwatts.

EPR spectra of fusion proteins were also collected. The spectra

were normalized by double integration.

Circular Dichroism (CD)

—CD measurements of monomeric

fusion protein were performed at a concentration of 5

buffer (10 m

phosphate with no salt, pH 7.4). CD spectra of

fibrils were obtained at 30

in the same phosphate buffer.

The CD spectra were obtained using a Jasco 815 spectropo-

larimeter (Jasco, Inc., Easton, MD). Measurements were taken

every 0.5 nm at a scan rate of 50 nm/min with average time of

1 s. Approximately 20–30 scans were averaged between 190

and 260 nm, and buffer backgrounds were subtracted. Spectra

were analyzed using the DichroWeb suite of programs (23)

including CDSSTR (24–26), SELCON3 (27), K2D (28, 29), and

CONTINLL (30, 31).

Temperature dependence of the CD spectra was measured

using a Jasco PFD-425s temperature controller. HDx1 fibrils

were incubated at a given target temperature for at least 20 min

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prior to recording the CD spectra. All other experimental

details remained the same as described above. In the tempera-

ture range between 0 and 50 ºC, spectral changes were 95–100%

reversible. The difference spectrum for 50 ºC and 0 ºC was

smoothed using the Savitzky-Golay method available on the

Jasco spectrometer.

RESULTS

To investigate the structural changes that occur in 46Q-con-

taining HDx1 upon aggregation, we generated 17 different

derivatives (Fig. 1

) that contained the spin-labeled side chain

R1 (Fig. 1

). We examined the derivatives by EPR in their sol-

uble and fibrillar forms. Due to its better solubility, the HDx1-

thioredoxin fusion protein was used to study the soluble form

whereas fibrils were formed from HDx1 in which the thiore-

doxin partner had been cleaved off. In agreement with previous

studies (10, 32, 33), we find that HDx1 forms predominantly

relatively short fibrils (100–500-nm length, 10–12-nm diame-

ter) that have a tendency to associate laterally and form bundles

(Fig. 1

Soluble HDx1 as a Fusion Protein Is Highly Dynamic

—To

ensure that the native protein structure was not perturbed by

the addition of the spin label, we compared CD spectra of the

soluble fusion protein with and without label and found that

they did not differ significantly (

supplemental Fig. S1

As illustrated in Fig. 2, the EPR spectra of all spin-labeled,

soluble HDx1-thioredoxin fusion proteins consist of three

sharp and narrowly spaced lines typical of highly mobile sites.

Given the relatively large size of the fusion protein (28 kDa), this

high mobility cannot solely be caused by rapid tumbling of the

protein. Rather, the data indicate a highly dynamic structure

consistent with a previous study using CD and NMR that found

unpolymerized HDx1-thioredoxin fusion protein to be largely

disordered (34). Although all of our spectra indicate that solu-

ble HDx1 is highly dynamic, there are subtle differences in the

sharpness (

i.e.

spin label motion) of the spectra. The widths of

the central resonance line for sites within the first 30 amino

acids are consistently larger (1.8–2.0 gauss) than those of sub-

sequent sites (1.6–1.75 gauss). These data indicate that the first

30 amino acids are less mobile than the latter amino acids.

Tethering of the HDx1 to the fusion partner could cause some

of this reduction in mobility. Inasmuch as tethering typically

affects the mobility of just the first 5–10 amino acids (35, 36),

residual secondary is likely to be present. This notion is consist-

ent with computational work suggesting that the N terminus of

HDx1 is likely to be partially ordered (17).

HDx1 Fibril Formation and CD Analysis

—To obtain uniform

fibrils for the different spin-labeled derivatives, we seeded each

fibril reaction with the same batch of native HDx1 seeds. Such

seeding has previously been shown to faithfully propagate the

biophysical properties of HDx1 through multiple generations

(37). Spin-labeled HDx1 grew readily from native fibril seeds,

indicating that the derivatives took up native structure. In addi-

tion, CD spectra of fibrils formed from several derivatives were

compared and found to be highly similar (

supplemental

Fig. S2

The CD spectra were fitted using the DichroWeb (23) suite of

fitting programs. All of these programs indicated that HDx1

FIGURE 1.

Spin labeling and fibril formation of HDx1 with 46Q.

, spin

labels introduced at the indicated sites in different HDx1 domains.

Colors

highlight the indicated domains.

, structure of spin-labeled side chain R1.

, representative negative stain electron micrograph of spin-labeled HDx1,

labeled at position 64.

Scale bar

is 100 nm.

FIGURE 2.

EPR spectra of the thioredoxin-HDx1 fusion protein indicate a

highly dynamic structure prior to aggregation.

X-band EPR spectra were

collected at 150-gauss scan width at room temperature.

Numbers

indicate

locations of the spin label in the amino acid sequences of HDx1 containing 46

Gln. For better visualization, all spectra are shown at the same amplitude.

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fibrils contain

35–45%

-sheet structure and

5–10%

-hel-

ical structure. These data are consistent with previous findings

which suggest the presence of

-sheet structure in the polyQ

region and

-helical structure in the N terminus (21). 30–50%

of the structure was fit to be disordered. It should be noted,

however, that these programs do not distinguish between poly-

proline II helices and disordered structure, which result in sim-

ilar CD spectra (see below).

The PolyQ Domain Becomes Highly Ordered in Fibrils, but It

Does Not Have a Parallel, in-Register Structure

—Upon fibril

formation, the most significant changes in the EPR spectra are

within the polyQ domain (Fig. 3,

red spectra

). All sites in this

domain display significant line broadening and increased sepa-

ration of the outer peaks, indicating that the polyQ domain

becomes strongly immobilized upon aggregation. Such immo-

bilization is typical for core regions of all amyloid fibrils that

have been investigated by EPR (38). What is different about the

spectra for HDx1 fibrils, however, is that no site in any region of

the protein displays the highly characteristic, single-line EPR

spectra that have been seen in

-synuclein, tau,

2m, human

prion protein, and IAPP (38–41). Single-line EPR spectra are

the consequence of the spin exchange that occurs in a parallel,

in-register structure in which four or more spin labels stack on

top of another and come into physical contact (38, 39). Thus,

unlike other amyloid fibrils, the HDx1 fibrils formed in this

study do not have a parallel, in-register structure in which each

protein takes up a new layer along the fibril axis, and the same

sites come into close contact with one another.

To test whether any spin-spin interaction could be detected

between the same labeled residues, we performed a dilution

experiment in which fibrils were grown from a mixture of 10%

labeled and 90% unlabeled protein. By reducing the density of

spin-labeled proteins, any effects of spin-spin interaction

become strongly diminished. The spectra of diluted HDx1

fibrils are qualitatively similar to those of the fully labeled

forms, but spin dilution causes an increase in the spectral

amplitude at most sites, including the polyQ domain (Fig. 3,

blue spectra

). Such spectral changes only occur when labels

come within 20 Å of each other (42). Thus, although sites in the

polyQ are not parallel and in-register, they are still within 20 Å

of each other in the fibril. This might be expected if the polyQ

domains of different molecules interact with each other. These

data also indicate the spin-labeled and native, unlabeled HDx1

can co-mix in fibrils and take up equivalent structures.

HDx1 C Terminus Lies Outside the Fibril Core

—All of our

C-terminal sites have three, easily discernible, relatively sharp

resonance lines upon fibril formation (Fig. 3). Hence, these sites

have elevated mobility and do not engage in significant tertiary

or quaternary contacts. Despite the overall high mobility, there

is a notable increase in mobility within this domain toward the

extreme C terminus. These data indicate that the C terminus is

anchored at the polyQ domain, from where it becomes increas-

ingly dynamic. In agreement with this notion, the spin-spin

interactions become weaker toward the C terminus and

become barely detectable at residues 76 and 111 (Fig. 3). Thus,

especially in the most C terminus, same residues are mostly

separated by more than 20 Å.

The transition from a highly immobilized polyQ domain to

the more dynamic C-terminal domain can also be seen in the

spectra of 63R1 and 64R1, which are at the border between the

polyQ and C-terminal domains. Both spectra exhibit a mobile

(see

arrows

in Fig. 3) as well as a strongly immobilized compo-

nent, suggesting that they have structural features of both

domains. Moreover, the C terminus seems to be capable of

locally loosening up the packing in the adjacent polyQ domain.

Polyproline II Structure in HDx1 Fibrils

—Despite the high

overall mobility, the EPR spectra of C-terminal sites reveal a

gradual increase in mobility spanning a stretch of about 40

amino acids (Fig. 3, also see below). This behavior could be due

to residual polyproline II structure. The highly proline-rich C

terminus has been predicted (21, 43, 44), but not shown

directly, to take up such a structure in HDx1 fibrils.

To test whether polyproline II structure might be present in

fibrils, we recorded CD spectra at different temperatures. It is

well established that polyproline II structures become more

ordered upon cooling. This ordering is typically very gradual

and occurs over a broad temperature range between 100 and

0 ºC (45, 46). Although heating HDx1 fibrils to higher temper-

atures caused aggregation, we found that it was possible to

record CD spectra of HDx1 fibrils between 0 and 50 ºC without

causing irreversible changes to the sample. The spectra

obtained at 0 and 50 ºC are given in Fig. 4

. The corresponding

difference spectrum (Fig. 4

) has the typical features of a poly-

proline II structure including a minimum at 206 nm and a max-

imum near 224 nm (47). As commonly observed for polyproline

II (45, 47), both spectral features increased with decreasing

temperature. It is important to note that the opposite trend

would have been observed for the unfolding of

-helical or

-sheet structures into a random coil. To test whether the for-

mation of polyproline II exhibits the typical gradual tempera-

ture transition (45, 46), we systematically varied the tempera-

ture and monitored the mean residue ellipticity at 206 nm. As

expected, the amount of polyproline II structure changed

FIGURE 3.

EPR spectra of spin-labeled HDx1 fibrils.

100% (

red

) and 10%

(

blue

) labeled fibrils for each site are overlaid. All spectra of 10% labeled fibrils

are shown at the same amplitude with respect to each other. To illustrate the

effects of spin-spin interaction, the corresponding pairs of spectra at 10 and

100%labelingareshownnormalizedtothesamenumberofspins.The

arrows

denote a mobile component in the last residue of the polyQ stretch as well as

the first residue in the polyproline region. X-band EPR spectra were recorded

at 150-gauss scan width at room temperature. The

numbers

indicate the loca-

tions of the spin label in the amino acid sequences of HDx1 containing 46 Gln.

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almost linearly without exhibiting a defined folding/unfolding

transition (Fig. 4

It is generally difficult to quantify the amount of polyproline

II structure, as the reference spectra for the fully folded and

unfolded states cannot readily be obtained (45). This is partic-

ularly difficult in the present study, as we presumably cannot

fully denature the polyproline II structure at 50 ºC. Thus, only

rather approximate estimates of polyproline II structure can be

given in the present case. Prior studies on polyproline model

peptides found that the mean residue ellipticity for the 224 nm

peak changed by about 1000 ºC cm

dmol

as the temperature

was varied between 0 and 50 ºC (45, 46). Here, we observe about

a quarter of that value suggesting that about a quarter (

20–30

residues) in HDx1 fibrils might be able to take up polyproline II

structure.

The N Terminus Becomes Immobilized in the Fibrils

—A

recent solid-state NMR study found that the HDx1 N terminus

in fibrils takes up an

-helical structure (21). In agreement with

this predicted structural ordering, the EPR spectra of N-termi-

nal sites exhibit significant immobilization (Fig. 3). However,

the immobilization exceeds that typically observed on the sur-

face of an

-helix, indicating the presence of tertiary and qua-

ternary contacts on both hydrophobic (residues 3 and 11) and

hydrophilic (residues 5 and 9) faces. There are also significant

spin-spin interactions for each of these sites, suggesting that the

N termini from different molecules come into proximity.

DISCUSSION

In the present study we define the domain organization of

mutant HDx1 in soluble and fibrillar form in terms of mobility

and intermolecular proximity. To summarize the mobility

information contained in the data, we used the semiquantita-

tive mobility parameter, the inverse of the width of the central

resonance line,

(48). As in previous studies (40, 41, 49),

this analysis was performed on the spectra from the spin-di-

luted fibrils to minimize any effects on line broadening due to

spin-spin interaction. Although the soluble protein is rather

dynamic, significant immobilization can be seen upon fibril for-

mation. This is especially the case for the N terminus and the

polyQ domain (Fig. 5

The polyQ domain has long been thought to form the amy-

loid core. As expected, we found that this domain is tightly

packed, but there are significant differences in the motilities at

its N and C termini. These differences in mobility depend on

the nature of the flanking domain. The N terminus, which is

almost as immobilized as the polyQ domain, has previously

been shown to take up an

-helical structure (21). Our data are

consistent with a structuring of this domain. In addition, we

find that this domain makes tertiary or quaternary contacts.

According to the detectable spin-spin interaction, these con-

tacts are very likely with other N termini. This notion is con-

sistent with cross-linking data (50) as well as a recent analytical

ultracentrifugation study demonstrating that N termini form

-helix-rich cores of oligomers (19). Thus, the N terminus is

not only important for oligomer formation but it also stabilizes

the fibril via its packing interactions, and it must be buried

within the fibril (Fig. 5

). The present study did not determine

whether the N terminus is located at the periphery or the center

of the core region.

Compared with the N terminus, the C terminus is much

more mobile. This mobility gradually increases with increasing

distance from the polyQ core domain. Although the C terminus

is very dynamic, with no tertiary or quaternary interactions, the

increase in mobility occurs gradually such that the mobility

does not reach that of the soluble fusion protein until about

residue 111 (Fig. 5

). This increase in mobility, which occurs

over more than 40 amino acids, again suggests the presence of a

residual secondary structure, rather than merely a tethering

effect. Our CD data indicate a polyproline II helical structure in

FIGURE 4.

TemperaturedependenceofHDx1fibrilsindicatespolyproline

II structure.

, CD spectra of HDx1 fibrils (30

)at0°C(

solid line

) and 50 °C

(

dashed line

) shown using mean residue ellipticity.

, difference spectrum for

the spectra of

, mean residue ellipticity values at 206 nm for CD spectra of

HDx1 obtained at different temperatures.

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HDx1 fibrils. Given that the N terminus and the polyQ region

are devoid of proline residues whereas the C terminus is rich in

proline (

60%), we propose that the C terminus adopts a poly-

proline II structure in HDx1 fibrils. This polyproline II struc-

ture lies outside the fibril core; it does not make any significant

intermolecular contacts and must be largely solvated (Fig. 5

In contrast to the N terminus, the C terminus does not engage

in contacts that could promote fibril stability. Rather, it appears

to destabilize the adjacent glutamine residues. Gln-63 is the

final glutamine of the polyQ domain, and Pro-64 is the first

proline residue of the proline-rich C terminus. Both residues

reside in a region of intermediate mobility, and their spectra

reflect the properties both of the polyQ as and the C-terminal

domains. Consistent with these structural data, biochemical

experiments show that the C-terminal proline stretches inhibit

aggregation of polyQ (51) and HDx1 (16, 22, 44). A

potential

role of the polyproline domain in influencing the local struc-

ture of the polyQ domain is further supported by a recent

crystallography study of soluble wild-type HDx1, which

found that the four glutamines adjacent to the proline

stretch are in an extended, rather than a random coil confor-

mation (52). Our data may offer insight into the observation

that MW7, an antibody that binds the polyproline region

immediately adjacent to the polyQ domain (53), is capable of

disaggregating preformed fibrils (54). Although further

studies will be required to map and characterize the precise

interactions of antibodies with the fibrils, epitopes in this

region may be especially promising targets for therapies

designed to reduce aggregation. It is accessible to the anti-

body and binding may enhance the naturally destabilizing

effect of the C-terminal tail.

Whereas the mobility profile described here has many simi-

larities to those of amyloid fibrils, the spin-spin interactions

contained in the EPR spectra of HDx1 fibrils are different from

those of previously described, disease-related amyloid fibrils.

No regions of the HDx1 fibrils display detectable evidence of

spin exchange, indicating that there is no stacking of multiple

residues in a parallel, in-register arrangement of

-strands (Fig.

). Although unusual, because nearly all reported fibrils

with amyloid cores over 20 amino acids in length have a

parallel, in-register

-strand arrangement, this is not neces-

sarily surprising. Many proteins may prefer the latter

arrangement because it maximizes hydrophobic contacts by

allowing like hydrophobic residues to stack on top of one

another (38). The homopolymer nature of polyQ could make

it more difficult for identical sites in the primary sequence to

find each other and stack in this manner. Thus, although the

commonly seen parallel, in-register structure is inconsistent

with the present data, we cannot exclude a more loosely

arranged parallel structure in which each molecule takes up

more than one layer (Fig. 6

), residues are not perfectly in-

), or there is some combination of the two.

Our data are also consistent with an antiparallel

-sheet

structure (55–57) (Fig. 6

We have begun to elucidate the structural features of the

various domains in HDx1 fibrils in the biologically relevant full-

FIGURE 5.

, inverse central line width from EPR spectra of HDx1 fusion pro-

tein (

circles

) and sparsely labeled fibrils (

triangles

). The inverse of the width of

the central resonance line is plotted as function of residue number to give a

measure of mobility. Individual domains are

highlighted

. The

orange

blue

and

green shaded regions

represent the N-terminal, polyQ, and C-terminal

domains, respectively.

, sketch of the domain organization within HDx1

fibrils. The N termini (

orange

) and polyQ (

blue

) form the fibril core region (

gray

cylinder

), from which the solvated, highly mobile C termini (

green

) protrude.

The polyQ region is represented as generic

stacked rectangles

and is not

meant to imply any statement about the actual number or arrangement of

individual

-strands.WhethertheN-terminalregionislocatedatthecenteror

the periphery of the core region is unknown.

FIGURE 6.

Sketches of different types of cross-

structures.

, HDx1 does

not adopt a parallel, in-register structure common to other disease-causing

amyloid proteins, in which multiple spin labels (

yellow dots

) come into close

contact.

,onepotentialstructureinwhichspinlabelsapproachwithin20Åis

a parallel arrangement of

-strands (

arrows

) in which each molecule takes up

morethanonelayerandspinlabelsareseparatedbyoneormoreintervening

layers of

-strands.

, another possibility would be a parallel structure in

which each molecule takes up a separate layer, but, due to the promiscuous

nature of polyQ, labels are not in register.

, alternatively, several antiparallel

models are possible, with or without intervening

-strands. The fibril axis is

indicated,whichisalsothedirectionofmainchainhydrogenbonds.Different

shades

arrows

represent

-strands from different HDx1 molecules.

Structure and Domain Organization of Huntingtin Fibrils

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length version of the protein. Together with a recent solid-state

NMR study, our findings represent a first step toward a high

resolution structure of HDx1 fibrils. Future EPR experiments

using a more exhaustive set of distance constraints from single-

and double-labeled mutants should allow us to obtain a more

precise idea of the arrangement of glutamine strands, as well as

the orientations of the N and C termini. Recent spin-labeling

work, which combined continuous wave EPR, pulsed EPR, and

computational refinement, demonstrated that detailed, three-

dimensional structural information can be obtained using such

an approach (58, 59).

Acknowledgments—We thank Konstantin Piatkov for advice regard-

ing protein purification and Ansgar Siemer for helpful discussion.

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